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the primary objective of the collaboration was to examine aspects of screening for type 2 diabetes and impaired glucose tolerance across various populations and ethnic groups . , studies included in the detect-2 database , in which retinopathy data had been collected , were invited to provide these data for this analysis . additional studies with retinopathy data identified by coinvestigators through personal contact or literature search also were invited to contribute datasets . this analysis focuses on nine studies from five countries that had retinopathy data by grading . participants aged 2079 years , including those with known diabetes and with gradable retinal photographs and at least one measure of glycemia , were included . all studies were approved by respective institutional review boards and were conducted according to the declaration of helsinki . retinopathy was classified as present or absent for initial analysis . where data were available , those with retinopathy were further classified as those having minimal nonproliferative diabetic retinopathy ( npdr ) , mild npdr , moderate npdr , severe npdr , or proliferative diabetic retinopathy ( pdr ) based on the information provided by individual studies using the modified airlie house classification levels ( 19 ) , modified early treatment diabetic retinopathy study levels ( 20 ) , or the fukuda standard ( 21 ) . levels 1420 indicate minimal npdr , levels 3035 or the fukuda standard a1 indicate mild npdr , levels 4047 or the fukuda standard a2 indicate moderate npdr , levels 5053 or the fukuda standard a3 indicate severe npdr , and levels 6090 or the fukuda standards a4 and b1b4 indicate pdr . the final retinopathy grading for each participant was based on the diagnosis in the more severely affected eye . the primary outcome used in this study was diabetes - specific retinopathy , which we defined as moderate or more severe levels of retinopathy . all nine studies measured plasma glucose , and six studies that measured a1c used high - performance liquid chromatography , of which five used a diabetes control and complications trial ( dcct)-aligned assay ( 5,12,14,15,18 ) . prevalence of diabetes - specific retinopathy was examined by 1 ) 0.5-unit intervals of glycemic measures and 2 ) vigintiles ( dividing participants into 20 equally sized groups ) of the distribution for each measure of glycemia . logistic regression models were applied to test the relationships between diabetes - specific retinopathy and glycemia by 0.5-unit intervals and by vigintiles of each glycemic measure , with the lowest range as the reference . the discriminatory power of each measure of glycemia for retinopathy was assessed as the area under the receiver - operating characteristic ( roc ) curve ( auc ) . an auc of 1 indicates perfect discriminatory power and an auc of 0.5 indicates that the discrimination is no better than chance . the impact of various thresholds on the prevalence of diabetes was examined by applying these values to 16,381 participants without known diabetes who had all three measures of glycemia . sensitivity analysis was performed on 1 ) studies in which a dcct - aligned assay for a1c was used ( ausdiab , chennai urban rural epidemiological study [ cures ] , multi - ethnic study of atherosclerosis and air pollution [ mesa ] , nhanes iii , and singapore malay eye study [ simes ] ) ; 2 ) studies in which one of the authors ( t.y.w ) was personally involved in the grading of retinopathy using the modified early treatment diabetic retinopathy study ( atherosclerosis risk in communities [ aric ] , ausdiab , blue mountains eye study [ bmes ] , mesa , and simes ) ; 3 ) studies in which participants were predominantly caucasian ( aric , ausdiab , bmes , mesa , and nhanes iii ) ; 4 ) studies in which participants were asian ( cures , hiroshima study , and simes ) ; and 5 ) studies in which participants had all three measures of glycemia . all statistical analyses were performed using sas 9.1 for windows ( sas institute , cary , nc ) and spss 16.0 for windows ( spss , chicago , il ) . retinopathy was classified as present or absent for initial analysis . where data were available , those with retinopathy were further classified as those having minimal nonproliferative diabetic retinopathy ( npdr ) , mild npdr , moderate npdr , severe npdr , or proliferative diabetic retinopathy ( pdr ) based on the information provided by individual studies using the modified airlie house classification levels ( 19 ) , modified early treatment diabetic retinopathy study levels ( 20 ) , or the fukuda standard ( 21 ) . levels 1420 indicate minimal npdr , levels 3035 or the fukuda standard a1 indicate mild npdr , levels 4047 or the fukuda standard a2 indicate moderate npdr , levels 5053 or the fukuda standard a3 indicate severe npdr , and levels 6090 or the fukuda standards a4 and b1b4 indicate pdr . the final retinopathy grading for each participant was based on the diagnosis in the more severely affected eye . the primary outcome used in this study was diabetes - specific retinopathy , which we defined as moderate or more severe levels of retinopathy . all nine studies measured plasma glucose , and six studies that measured a1c used high - performance liquid chromatography , of which five used a diabetes control and complications trial ( dcct)-aligned assay ( 5,12,14,15,18 ) . prevalence of diabetes - specific retinopathy was examined by 1 ) 0.5-unit intervals of glycemic measures and 2 ) vigintiles ( dividing participants into 20 equally sized groups ) of the distribution for each measure of glycemia . logistic regression models were applied to test the relationships between diabetes - specific retinopathy and glycemia by 0.5-unit intervals and by vigintiles of each glycemic measure , with the lowest range as the reference . the discriminatory power of each measure of glycemia for retinopathy was assessed as the area under the receiver - operating characteristic ( roc ) curve ( auc ) . an auc of 1 indicates perfect discriminatory power and an auc of 0.5 indicates that the discrimination is no better than chance . the impact of various thresholds on the prevalence of diabetes was examined by applying these values to 16,381 participants without known diabetes who had all three measures of glycemia . sensitivity analysis was performed on 1 ) studies in which a dcct - aligned assay for a1c was used ( ausdiab , chennai urban rural epidemiological study [ cures ] , multi - ethnic study of atherosclerosis and air pollution [ mesa ] , nhanes iii , and singapore malay eye study [ simes ] ) ; 2 ) studies in which one of the authors ( t.y.w ) was personally involved in the grading of retinopathy using the modified early treatment diabetic retinopathy study ( atherosclerosis risk in communities [ aric ] , ausdiab , blue mountains eye study [ bmes ] , mesa , and simes ) ; 3 ) studies in which participants were predominantly caucasian ( aric , ausdiab , bmes , mesa , and nhanes iii ) ; 4 ) studies in which participants were asian ( cures , hiroshima study , and simes ) ; and 5 ) studies in which participants had all three measures of glycemia . all statistical analyses were performed using sas 9.1 for windows ( sas institute , cary , nc ) and spss 16.0 for windows ( spss , chicago , il ) . in total , 44,623 participants had information on both the presence and severity of retinopathy ( table 1 ) . a total of 1,589 participants had minimal npdr , 762 had mild npdr , 430 had moderate npdr , 50 had severe npdr , and 171 had pdr . the number of participants available for each measure of glycemia was 41,334 for fpg , 21,334 for 2-h pg , and 27,933 for a1c . of these , 27,445 participants had at least two measures and 18,533 participants had all three measures . the characteristics of participants by study are shown in supplementary table 1 in the online appendix ( available at http://care.diabetesjournals.org/cgi/content/full/dc10-1206/dc1 ) . summary of studies included in these analyses * number of participants aged 2079 years included in the analysis . the overall prevalence of any retinopathy was 6.7% and 1.5% for diabetes - specific retinopathy . in people with known diabetes , the prevalence of diabetes - specific retinopathy was 9.4% , in newly diagnosed diabetes 1.0% , in impaired glucose tolerance ( 1 ) 0.1% , in impaired fasting glucose ( 1 ) 0.1% , and with normal glucose tolerance 0.1% . figure 1 shows the prevalence of retinopathy by 0.5-unit intervals for each measure of glycemia for diabetes - specific retinopathy . diabetes - specific retinopathy was virtually absent ( prevalence < 0.4% ) at low levels for each glycemic measure but began to increase from the fpg category of 6.06.4 the curve for 2-h pg was flatter than for fpg and a1c , and no definite interval of increase for 2-h pg was obvious . prevalence of diabetes - specific retinopathy ( moderate or more severe retinopathy ) with 95% confidence intervals , number of retinopathy cases , and participants within each interval by 0.5 unit intervals for fpg and 2-h pg , and a1c . logistic regression adjusted for study center showed that the first interval where the odds ratio ( or ) for diabetes - specific retinopathy was significantly different from the reference fpg level of 4.04.4 mmol / l was 6.56.9 mmol / l ( or 6.0 [ 95% ci 2.117.1 ] ; p < 0.01 ) . the corresponding result for a1c was 6.56.9% ( 16.8 [ 2.3123.7 ] ; p = 0.01 ) compared with an a1c of 4.04.4% . figure 2 shows the prevalence of diabetes - specific retinopathy by vigintiles of the glycemic distributions . the prevalence of diabetes - specific retinopathy was very low until the 15th vigintile for 2-h pg ( vigintile range 9.810.6 mmol / l ) and until the 17th vigintile for fpg ( 6.46.8 mmol / l ) and for a1c ( 6.16.2% ) . prevalence of diabetes - specific retinopathy ( moderate or more severe retinopathy ) by vigintiles of the distribution of fpg , 2-h pg , and a1c . logistic regression models adjusted for study center confirmed a statistically significant difference in the or for diabetes - specific retinopathy compared with the first vigintile that occurred from the 15th vigintile for 2-h pg ( vigintile range 9.810.6 mmol / l ; or 10.1 [ 95% ci 1.379.4 ] ; p = 0.03 ) , from the 17th vigintile for fpg ( 6.46.8 mmol / l ; 2.5 [ 1.25.2 ] ; p = 0.01 ) , and from the 18th vigintile for a1c ( 6.36.7% ; 4.5 [ 1.415.2 ] ; p = 0.01 ) . the overall discriminatory power determined by aucs was uniformly high for diabetes - specific retinopathy for each measure of glycemia ( 0.87 [ 95% ci 0.850.89 ] for fpg , 0.89 [ 0.870.91 ] for 2-h pg , and 0.90 [ 0.880.92 ] for a1c ) . the overlapping cis suggests that there is no statistical difference between the three measures of glycemia . the performance of a wide range of thresholds was examined , with particular attention to those that overlapped from the continuous and vigintile distribution plots . mmol / l for fpg , 13.0 mmol / l for 2-h pg , and 6.4% for a1c ( table 2 ) . if these thresholds were used for diagnosing diabetes , the prevalence of newly diagnosed diabetes would be 11.9 , 8.0 , and 6.3% according to fpg , 2-h pg , and a1c , respectively . the differences in performance based on roc curve statistics for the three measures of glycemia were minor for threshold values around the above values ( supplementary table 2 ) . threshold ranges for diabetes - specific retinopathy ( moderate npdr or more severe retinopathy ) derived from logistic regression models ( adjusted for center ) of the glycemic measures by continuous distribution and vigintile distribution and roc curve analysis sensitivity analyses showed that the five studies in which t.y.w used the same retinopathy grading system or the five studies that used dcct - aligned assays for a1c measurements provided similar results to the overall study . in total , 44,623 participants had information on both the presence and severity of retinopathy ( table 1 ) . a total of 1,589 participants had minimal npdr , 762 had mild npdr , 430 had moderate npdr , 50 had severe npdr , and 171 had pdr . the number of participants available for each measure of glycemia was 41,334 for fpg , 21,334 for 2-h pg , and 27,933 for a1c . of these , 27,445 participants had at least two measures and 18,533 participants had all three measures . the characteristics of participants by study are shown in supplementary table 1 in the online appendix ( available at http://care.diabetesjournals.org/cgi/content/full/dc10-1206/dc1 ) . summary of studies included in these analyses * number of participants aged 2079 years included in the analysis . the overall prevalence of any retinopathy was 6.7% and 1.5% for diabetes - specific retinopathy . in people with known diabetes , the prevalence of diabetes - specific retinopathy was 9.4% , in newly diagnosed diabetes 1.0% , in impaired glucose tolerance ( 1 ) 0.1% , in impaired fasting glucose ( 1 ) 0.1% , and with normal glucose tolerance 0.1% . figure 1 shows the prevalence of retinopathy by 0.5-unit intervals for each measure of glycemia for diabetes - specific retinopathy . diabetes - specific retinopathy was virtually absent ( prevalence < 0.4% ) at low levels for each glycemic measure but began to increase from the fpg category of 6.06.4 mmol / l and from the a1c category of 6.06.4% . the curve for 2-h pg was flatter than for fpg and a1c , and no definite interval of increase for 2-h pg was obvious . prevalence of diabetes - specific retinopathy ( moderate or more severe retinopathy ) with 95% confidence intervals , number of retinopathy cases , and participants within each interval by 0.5 unit intervals for fpg and 2-h pg , and a1c . logistic regression adjusted for study center showed that the first interval where the odds ratio ( or ) for diabetes - specific retinopathy was significantly different from the reference fpg level of 4.04.4 mmol / l was 6.56.9 mmol / l ( or 6.0 [ 95% ci 2.117.1 ] ; p < 0.01 ) . the corresponding result for a1c was 6.56.9% ( 16.8 [ 2.3123.7 ] ; p = 0.01 ) compared with an a1c of 4.04.4% . figure 2 shows the prevalence of diabetes - specific retinopathy by vigintiles of the glycemic distributions . the prevalence of diabetes - specific retinopathy was very low until the 15th vigintile for 2-h pg ( vigintile range 9.810.6 mmol / l ) and until the 17th vigintile for fpg ( 6.46.8 mmol / l ) and for a1c ( 6.16.2% ) . prevalence of diabetes - specific retinopathy ( moderate or more severe retinopathy ) by vigintiles of the distribution of fpg , 2-h pg , and a1c . logistic regression models adjusted for study center confirmed a statistically significant difference in the or for diabetes - specific retinopathy compared with the first vigintile that occurred from the 15th vigintile for 2-h pg ( vigintile range 9.810.6 mmol / l ; or 10.1 [ 95% ci 1.379.4 ] ; p = 0.03 ) , from the 17th vigintile for fpg ( 6.46.8 mmol / l ; 2.5 [ 1.25.2 ] ; p = 0.01 ) , and from the 18th vigintile for a1c ( 6.36.7% ; 4.5 [ 1.415.2 ] ; p = 0.01 ) . the overall discriminatory power determined by aucs was uniformly high for diabetes - specific retinopathy for each measure of glycemia ( 0.87 [ 95% ci 0.850.89 ] for fpg , 0.89 [ 0.870.91 ] for 2-h pg , and 0.90 [ 0.880.92 ] for a1c ) . the overlapping cis suggests that there is no statistical difference between the three measures of glycemia . the performance of a wide range of thresholds was examined , with particular attention to those that overlapped from the continuous and vigintile distribution plots . the thresholds that optimized sensitivity and specificity were 6.6 mmol / l for fpg , 13.0 mmol / l for 2-h pg , and 6.4% for a1c ( table 2 ) . if these thresholds were used for diagnosing diabetes , the prevalence of newly diagnosed diabetes would be 11.9 , 8.0 , and 6.3% according to fpg , 2-h pg , and a1c , respectively . the differences in performance based on roc curve statistics for the three measures of glycemia were minor for threshold values around the above values ( supplementary table 2 ) . threshold ranges for diabetes - specific retinopathy ( moderate npdr or more severe retinopathy ) derived from logistic regression models ( adjusted for center ) of the glycemic measures by continuous distribution and vigintile distribution and roc curve analysis sensitivity analyses showed that the five studies in which t.y.w used the same retinopathy grading system or the five studies that used dcct - aligned assays for a1c measurements provided similar results to the overall study . mmol / l , for a1c 6.46.5% , and for 2-h pg 10.111.2 mmol / l . the current diagnostic criteria for diabetes were derived from analyses of the relationship between retinopathy and measures of glycemia ( 1 ) . our study is the largest to examine this association , using data from 45,000 participants from five countries , and provides the statistical power for a more detailed and precise examination of glycemic thresholds for diabetes - specific retinopathy ( moderate nonproliferative and more severe retinopathy ) . previous studies ( 7,16 ) have only reported the association of glycemic measures with any retinopathy , which is less specific for hyperglycemia and is very frequently detected in people without diabetes . the association between glycemic measures and retinopathy has traditionally been investigated by plotting the prevalence of retinopathy against the decile distribution ( the population divided into 10 equal groups ) of each glycemic measure ( 1,2 ) . our large dataset allows analysis using vigintile distributions ( the population divided into 20 equal groups ) , which narrows the glycemic range of each group . based on logistic regression analysis of these vigintile distributions , glycemic thresholds for diabetes - specific retinopathy were observed in the range of 6.46.8 mmol / l for fpg , 9.810.6 . the large size of this dataset enables diabetes - specific retinopathy to be plotted against measures of glycemia as a continuous variable . a curvilinear relationship was observed , especially for fpg and a1c , as opposed to the linear association observed between blood pressure and cardiovascular disease . diabetes - specific retinopathy was rare at low levels of glycemia but increased from a range of 6.06.4 mmol / l for fpg and 6.06.4% for a1c . a threshold for increasing retinopathy was less obvious for 2-h pg , probably related to the smaller number of study participants with this measure and diabetes - specific retinopathy . change point analyses , which were used previously in two population - based studies ( 22 ) , were applied to these curves in an attempt to identify statistically significant thresholds , but we were unable to demonstrate a clear threshold for any glycemic measure by this method . this could suggest that within the ranges of visually detected thresholds for the three measures , changes in the prevalence of diabetes - specific retinopathy remain somewhat linear . the continuous and vigintile plots provided a similar range of threshold values for fpg and a1c . roc curve analyses were then used to compare performance in relation to optimizing sensitivity and specificity of glycemic values in the range around these thresholds . these analyses suggest thresholds of 6.6 mmol / l for fpg and 6.4% for a1c . the results derived from the vigintile distribution , continuous plots , and roc curve analyses suggest cut point values of 6.5 mmol / l for fpg and 6.5% for a1c , which could be considered in deliberations on modifying the current diagnostic criteria for diabetes . the results for 2-h pg were too inconsistent to consider modifying the current diagnostic cut point of 11.1 mmol / l . it should be noted that these values do not result in equivalent estimates for prevalent diabetes . this has been an ongoing issue with the current diagnostic criteria , whereby using fpg alone or an oral glucose tolerance test to diagnose diabetes gives different diabetes prevalence ( 23 ) . from our data ( supplementary table 2 ) , lowering the fpg to 6.5 mmol / l would result in a diabetes prevalence of 13.0% based on fpg alone and 18.6% based on an oral glucose tolerance test using an fpg of 6.5 mmol / l or a 2-h pg of 11.1 the prevalence of diabetes defined by an a1c of 6.5% is considerably lower ( 5.7% ) . this discrepancy in prevalence may be problematic for epidemiological studies but is not necessarily a disadvantage for individual patient care . an a1c of 6.5% was associated with a higher sensitivity and specificity than an fpg of 6.5 mmol / l and a higher specificity than a 2-h pg of 11.1 mmol / l . in other words , fewer people would be identified as having diabetes , but this would not compromise the identification of people with diabetes - specific retinopathy . whether this would have any deleterious ramifications in relation to identifying individuals at increased risk of other microvascular or macrovascular disease remains to be determined . there have been reports of differences in a1c levels independent of glucose between black , white , and south asian populations ( 24,25 ) . in our study , subgroup analysis by asian and predominantly caucasian populations showed no difference in the optimal a1c threshold ( 6.4% for both ) . however , our study was not designed to have and did not have sufficient numbers to examine a potential black / white difference . strengths of this study include its large sample size , which was drawn from populations across different countries and racial / ethnic groups ; the ability to focus on diabetes - specific retinopathy ; and availability of data to examine three glycemic measures . first , this study was based on cross - sectional data , whereas diagnostic thresholds would ideally be informed by incidence data of diabetes complications . second , the methods used to assess and classify retinopathy differed between studies , and it was not possible to independently review the grading of all photographs . nevertheless , inter- and intraobserver consistency for retinopathy in the different studies was of the order of 8098% ( 3,10,15 ) and misclassification , especially for moderate or more severe forms of retinopathy , is likely to be minimal but can not be entirely eliminated . furthermore , analysis of the studies in which t.y.w . was involved in the standardized grading of retinal photographs showed cut points for fpg and a1c similar to our entire study cohort . third , no quality assurance of measures of glycemia could be applied across the studies . nevertheless , all studies measured a1c using high - performance liquid chromatography , and analysis of the five studies that used a dcct - aligned assay showed an a1c cut point of 6.46.5% . fourth , the hiroshima study , with its large sample size , and the pima indian study , with its high prevalence of diabetes - specific retinopathy , may have influenced the results . however , sensitivity analyses that excluded these two studies did not alter the overall results . finally , not all included studies were randomly sampled populations ( e.g. , mesa ) and some ( e.g. , ausdiab ) oversampled people with diabetes and/or prediabetes . common to all such analyses is the issue of whether to include people with previously diagnosed diabetes . if people with known diabetes currently receiving blood glucose lowering treatment are included , the population - based characteristics of the study sample are maintained , but a bias associated with treatment - induced effects on glycemia is introduced and the level of glycemia assessed in each study may be lower than that which led to retinopathy . excluding people with treated diabetes from the analyses eliminates this bias but changes the characteristics of the population by eliminating many individuals with retinopathy , making it much more difficult to identify a threshold ( 2,7 ) . large incidence studies are needed to resolve these issues and determine the optimal levels of glycemia that predict the development of diabetes - specific retinopathy . in summary , this pooled analysis of glycemia and diabetes - specific retinopathy among close to 45,000 participants substantially broadens the evidence based on glucose - specific and a1c diabetes diagnostic thresholds . our results demonstrate narrow glycemic threshold ranges for the presence of diabetes - specific retinopathy and suggest that the current diabetes diagnostic level for fpg should be lowered to 6.5 mmol / l and that an a1c of 6.5% is a suitable alternative diagnostic criterion . the medical records of 227 patients diagnosed with retinoblastoma at seoul national university children 's hospital between march 1997 and september 2007 were retrospectively studied . the diagnosis of retinoblastoma was based on indirect ophthalmoscopy , computed tomography , and ultrasonography findings . the stage was assessed with r - e classification and the international classification of retinoblastoma ( icr ) . upon diagnosis of retinoblastoma , a brain magnetic resonance imaging and bone scan were performed to evaluate systemic metastasis . of the patients diagnosed with retinoblastoma , patients who had been treated with primary chemotherapy with or without the combination of various local therapeutic methods were selected and included in this study . patients whose tumors were small and able to be treated with local therapeutic methods alone were not included . patients with documented evidence of systemic metastasis or with evidence of extraocular extension at diagnosis were also excluded . patients who had chosen to undergo primary enucleation after thorough discussion of the available treatment options and their prognoses were also not included in this study . the chemotherapeutic regimen consisted of a 13-cycle combination of cisplatin ( carboplatin in some patients ) , etoposide , and vincristine , which is used in the treatment of central nervous system tumors [ 8,18 - 20 ] . an ophthalmoscopic examination was done either on an outpatient basis or under general anesthesia at the beginning of each cycle according to the patient 's condition . additionally , every patient was evaluated by a pediatric oncologist regularly for possible adverse effects . the chemotherapy regimen was changed to regimen 2 if the tumor showed a minimal response to the basic regimen or if adverse effects , such as nephrotoxicity , were detected . the chemotherapy regimen was changed to regimen 3 if the treatment response continued to be minimal and if growth of the tumor or new lesions were detected despite treatment either with the basic regimen or regimen 2 . the regimen was also modified according to the patients ' general condition and if any adverse effect of a specific chemotherapeutic agent was suspected . local therapy such as thermotherapy , cryotherapy , and laser photocoagulation was added depending on the status of each tumor after each cycle of chemotherapy . thermotherapy was done in patients with tumors with a base less than 3 mm and a thickness less than 3 mm without vitreous seeds located posterior to the equator . laser photocoagulation was done in similar cases , but also in slightly larger tumors by surrounding the tumor and closing off feeding vessels . the patients were grouped according to tumor classification ( r - e classification , icr ) , therapeutic methods used , and the status of the contralateral eye . enucleation was performed in eyes with tumors that showed persistent viable appearance with no signs of regression or in eyes with newly appearing lesions after two or more cycles of treatment . enucleation was also performed in eyes with media opacities rendering their tumors impossible to follow . the patients in which tumors had totally regressed and therefore avoided enucleation were considered to have had a favorable outcome . ebrt was not performed as a salvage treatment due to the superior efficacy of enucleation in tumor control and the possible occurrence of secondary malignancy and disfigurement of the orbit . informed consent was obtained from every patient at the time of diagnosis and initiation of treatment and also before the beginning of each cycle of chemotherapy . the chemotherapeutic regimen consisted of a 13-cycle combination of cisplatin ( carboplatin in some patients ) , etoposide , and vincristine , which is used in the treatment of central nervous system tumors [ 8,18 - 20 ] . an ophthalmoscopic examination was done either on an outpatient basis or under general anesthesia at the beginning of each cycle according to the patient 's condition . additionally , every patient was evaluated by a pediatric oncologist regularly for possible adverse effects . the chemotherapy regimen was changed to regimen 2 if the tumor showed a minimal response to the basic regimen or if adverse effects , such as nephrotoxicity , were detected . the chemotherapy regimen was changed to regimen 3 if the treatment response continued to be minimal and if growth of the tumor or new lesions were detected despite treatment either with the basic regimen or regimen 2 . the regimen was also modified according to the patients ' general condition and if any adverse effect of a specific chemotherapeutic agent was suspected . local therapy such as thermotherapy , cryotherapy , and laser photocoagulation was added depending on the status of each tumor after each cycle of chemotherapy . thermotherapy was done in patients with tumors with a base less than 3 mm and a thickness less than 3 mm without vitreous seeds located posterior to the equator . laser photocoagulation was done in similar cases , but also in slightly larger tumors by surrounding the tumor and closing off feeding vessels . the patients were grouped according to tumor classification ( r - e classification , icr ) , therapeutic methods used , and the status of the contralateral eye . enucleation was performed in eyes with tumors that showed persistent viable appearance with no signs of regression or in eyes with newly appearing lesions after two or more cycles of treatment . enucleation was also performed in eyes with media opacities rendering their tumors impossible to follow . the patients in which tumors had totally regressed and therefore avoided enucleation were considered to have had a favorable outcome . ebrt was not performed as a salvage treatment due to the superior efficacy of enucleation in tumor control and the possible occurrence of secondary malignancy and disfigurement of the orbit . informed consent was obtained from every patient at the time of diagnosis and initiation of treatment and also before the beginning of each cycle of chemotherapy . of the 227 patients diagnosed with retinoblastoma in our institution , a total of 52 patients , 65 eyes were included in this study . overall , 34 eyes achieved total regression of the tumor and were able to avoid enucleation . the probability of ocular survival was 46.566.70% using the kaplan - meier method ( fig . treatment outcomes of patients according to the r - e classification and icr are shown in table 3 . a relatively similar number of patients were in each group of r - e classification , but the majority of the eyes included in this study were in group d according to the icr . the majority of eyes with tumors of group c or lower were excluded from the study because they were eligible for local therapy alone . the eyes with group a and b tumors that were included in this study had undergone primary chemotherapy because of a group d tumor in the contralateral eye . there was a marked difference in the treatment outcome among patients classified as r - e group i to ii and r - e group iii to v. in patients with r - e classification i and ii tumors , complete remission of the tumors was observed in 100% and 86.7% of patients , respectively . in contrast , only 33.3% , 40% , and 20% of the r - e classification iii , iv , and v group patients avoided enucleation , respectively . in icr group the local modalities of treatment used in this study included cryotherapy , thermotherapy , and laser photocoagulation . twenty eyes in 18 patients received chemotherapy only , while 28 eyes in 21 patients were treated with chemotherapy and one modality of local therapy . seventeen eyes in 13 patients were treated with chemotherapy and two modalities of local therapy . the treatment outcome of patients with unilateral and bilateral involvement is shown in table 5 . three out of 23 eyes with unilateral involvement were saved while 13 out of the 16 eyes of patients with bilateral involvement who had undergone primary enucleation of one eye were saved . eighteen of the 26 eyes in patients with bilateral involvement and no enucleation were saved . tumors in the eyes with bilateral involvement and enucleation of the more severe eye had a significantly lower r - e classification ( p=0.001 ) . complications of the therapy included mild and manageable complications such as myelosupression , neutropenic fever , alopecia , mild nephrotoxicity , and ileus . the probability of overall survival was 96.773.17% using the kaplan - meier method ( fig . there was one case of hyphema and vitreous hemorrhage and one case of cataract , which was thought to be due to local therapy , but the direct relationship remains uncertain . in both cases enucleation primary chemotherapy , when combined with local therapy , showed a probability of ocular survival of 46.56% in retinoblastomas not treatable with local therapy alone . forty - three cases ( 66.2% ) in this study were group d or e tumors , and 16 of these 43 eyes ( 37.2% ) avoided enucleation . systemic chemoreduction followed by local therapy has shown various tumor control rates up to 78 to 85% . however , when comparing the treatment outcomes of these studies , the severity of the tumors in the subjects included in the studies should be taken into consideration . these studies usually have a heterogeneous severity of tumors included , often including a fair proportion of tumors of low severity . our study included only tumors that were considered otherwise untreatable with conventional treatment methods , which renders direct comparison of these previous survival rates with our results irrational . recently , successful tumor control has even been reported with chemotherapy alone in more advanced tumors . reported results of chemotherapy ( with six cycles of vincristine , etoposide , and carboblatin ) in group d heritable retinoblastoma . successful tumor control with chemotherapy was obtained in only 2 eyes ( 11% ) , although 9 more eyes ( 50% ) underwent successful salvage treatment with ebrt . shields et al . reported results of chemoreduction with 6 cycles of vincristine , etoposide , and carboplatin with or without prophylactic low - dose ebrt for group e retinoblastoma , with a globe salvage rate of 20/42 ( 48% ) in the chemoreduction only group and 4/5 ( 80% ) in the chemoreduction and prophylactic low - dose ebrt group . our results of a globe salvage rate of 37.2% in group d or e tumors without salvage treatment with ebrt is comparable to the results of previous studies . this treatment method can be considered an effective method with minimal complications ; it is especially valuable in bilateral cases when preservation of the eye and vision is more critical , or in cases when a patient strongly refuses enucleation . there was a tendency towards a better outcome in patients who were treated with more local treatment modalities . the salvage rate was 3/23 ( 13.0% ) in unilateral cases , compared to a salvage rate of 18/26 ( 69.3% ) in bilateral cases , indicating that there was a tendency towards enucleation in unilateral cases . this may have been due to a predilection to completely eradicate the tumor when even the slightest amount of evidence of progression was present . these findings suggest that with more vigorous treatment of the tumor with all possible additional local therapeutic methods , the overall eyeball salvage rate could be even higher . also , the local treatment modalities were selected according to the individual patient 's conditions and were not standardized . however , many of these shortcomings also apply to other studies regarding the treatment of retinoblastoma . in conclusion , chemoreduction and local therapy can be considered as a first - line treatment in low grade tumors . in higher grade tumors , although enucleation is still the treatment of choice in the most severe cases with vitreous seeding or massive tumors involving over half the retina , chemotherapy combined with local therapy can also be given as a choice of treatment to parents in intermediate cases , especially in bilateral cases with one eye already requiring enucleation . further study is necessary to determine the most effective chemotherapy regimen and local therapy protocol . the tendency of a better outcome in patients treated with more modalities of local therapy requires further study .

objectiveto re - evaluate the relationship between glycemia and diabetic retinopathy.research design and methodswe conducted a data - pooling analysis of nine studies from five countries with 44,623 participants aged 2079 years with gradable retinal photographs . the relationship between diabetes - specific retinopathy ( defined as moderate or more severe retinopathy ) and three glycemic measures ( fasting plasma glucose [ fpg ; n = 41,411 ] , 2-h post oral glucose load plasma glucose [ 2-h pg ; n = 21,334 ] , and a1c [ n = 28,010 ] ) was examined.resultswhen diabetes - specific retinopathy was plotted against continuous glycemic measures , a curvilinear relationship was observed for fpg and a1c . diabetes - specific retinopathy prevalence was low for fpg < 6.0 mmol / l and a1c < 6.0% but increased above these levels . based on vigintile ( 20 groups with equal numbers ) distributions , glycemic thresholds for diabetes - specific retinopathy were observed over the range of 6.46.8 mmol / l for fpg , 9.810.6 mmol / l for 2-h pg , and 6.36.7% for a1c . thresholds for diabetes - specific retinopathy from receiver - operating characteristic curve analyses were 6.6 mmol / l for fpg , 13.0 mmol / l for 2-h pg , and 6.4% for a1c.conclusionsthis study broadens the evidence based on diabetes diagnostic criteria . a narrow threshold range for diabetes - specific retinopathy was identified for fpg and a1c but not for 2-h pg . the combined analyses suggest that the current diabetes diagnostic level for fpg could be lowered to 6.5 mmol / l and that an a1c of 6.5% is a suitable alternative diagnostic criterion . purposeto evaluate the efficacy of primary chemotherapy combined with local therapy in the treatment of retinoblastomas not treatable with a single therapeutic method.methodswe performed a retrospective chart review of 227 patients diagnosed with retinoblastoma . sixty - five eyes in 52 patients had tumors not treatable with a single therapeutic method and received primary chemotherapy combined with local therapy as needed.resultstumor control and eye salvage was achieved in 34 of the 65 eyes ; the probability of ocular survival was 46.56% using the kaplan - meier method . forty - three of the 65 eyes were group d or e tumors , in which tumor control and eye salvage was achieved in 16 eyes . twenty eyes were treated with chemotherapy only , while 28 eyes received one additional modality of local therapy , and 17 eyes received two modalities of local therapy . of the eyes treated with chemotherapy only , tumor control was achieved in 5 eyes.conclusionsprimary chemotherapy combined with local therapy can be effective and safe in the treatment of retinoblastomas otherwise untreatable with other therapeutic methods , such as group d and e retinoblastomas . more vigorous treatment with more local therapeutic methods combined may yield even better results .

coal - tar pitch is an important feedstock of carbochemical origin used for the production of i.a . carbon anodes , graphite electrodes , fireproof materials , carbon - carbon composites and carbon fibres , as coking additive and as binder in many insulating and sealing materials used in construction and road building [ 1 , 2 ] . bitumens originating from coal , similarly as petroleum asphalts can be treated as colloidal - dispersive systems . micelles composed of 2 components and partly of 1 components form dispersed phase while oils ( components ) and partly components are the dispersing phase . because of the presence of carcinogenic aromatic hydrocarbons in coal - tar pitch , there is a need for its modification to obtain material safe for natural environment . research on the preparation of modified bituminous substances originating from coal , carried out for many years in the institute of chemistry of warsaw university of technology in plock has led to the elaboration of method reducing the level of carcinogenicity of coal - tar pitch . modification of coal - tar pitch with polymers influences also significantly its group composition and properties . the direction and scale of changes depend on chemical structure of the modifier and its amount . chemical structure is a factor directly determining properties of each of the components of bitumen - polymer compositions and simultaneously has an impact on the possibility to form a specific structure of a given composition , on which in turn its properties depend [ 59 ] . because of profitable properties of pitch - polymer mixtures , it would be advisable to check the possibilities to use waste polymers for the modification of the pitch . it leads to the search for new techniques and methods of evaluation of their homogenicity and application properties . one of them are microscopic studies , which play significant role in the determination of the structure and colloidal stability of bitumens and bitumen - polymer systems , including pitch - polymer compositions . in the studies of bitumen - polymer systems , fluorescence microscope is used , particularly for the evaluation of polymer - asphalt materials [ 1113 ] . however , there is no significant development in the evaluation of pitch - polymer compositions with the use of such microscope . the aim of this work was to determine the utility of fluorescence microscope for the evaluation of the structure of coal - tar pitch compositions with selected waste polymers and to study their influence on pitch properties . the raw materials used in this study were coal - tar pitch ( ctp ) and selected waste polymers : poly(ethylene terephthalate ) ( pet ) , poly(methylene methacrylate ) ( pmma ) and phenol - formaldehyde resin ( pf ) . pitch - polymer compositions containing from 10 to 50 wt% waste polymer were prepared in the conditions allowing to obtain homogeneous and stable mixtures . depending on the applied waste polymer , the components were homogenized in the temperatures from 150 to 270 c , during 0.52.5 h. the homogenization of the composition components was carried out at as low as possible temperatures ( from which it was possible to mix the components ) . idea was to eliminate of the destruction and/or degradation processes of waste polymers in coal - tar pitch during the preparation of compositions . the composition of mixtures and preparation conditions are presented in table 1.table 1preparation conditions and properties of pitch - polymer compositionscompositions(wt%)preparation of compositionssp(c)cv(wt%)ti(wt%)qi(wt%)ctp107.053.0334.217.1490 ctp + 10 pet260 c0.5 h127.053.6245.8013.3175 ctp + 25 pet166.050.1043.1029.4350 ctp + 50 pet236.038.0068.5062.7990 ctp + 10 pmma270 c1 h137.060.0835.628.7875 ctp + 25 pmma145.048.3329.369.2250 ctp + 50 pmma200.028.7923.0310.4990 ctp + 10 pf150 c2.5 h128.054.1655.1810.1475 ctp + 25 pf141.053.6763.044.4850 ctp + 50 pf*54.0788.544.21*infusible composition preparation conditions and properties of pitch - polymer compositions * infusible composition for coal - tar pitch and pitch - polymer compositions , the following measurements were carried out : softening point by ring and ball method ( sp ) according to the pn - en 1427:2009 standard , coking value ( cv ) according to the pn - c-97093:1993 standard , content of components insoluble in quinoline ( qi ) according to the pn - c-97058:1999 standard andcontent of components insoluble in toluene ( ti ) according to the method elaborated in the institute of chemistry , warsaw university of technology in plock . softening point by ring and ball method ( sp ) according to the pn - en 1427:2009 standard , coking value ( cv ) according to the pn - c-97093:1993 standard , content of components insoluble in quinoline ( qi ) according to the pn - c-97058:1999 standard and content of components insoluble in toluene ( ti ) according to the method elaborated in the institute of chemistry , warsaw university of technology in plock . the colloidal structure of pitch - polymer compositions by olympus bx41 microscope was carried out . in the study of colloidal structure of pitch - polymer compositions , in ultraviolet light , press moulding of compositions on the microscopic slide by a hydraulic press was on adopted technique of preparing of samples for study . parameters of press moulding of samples : pressure from 2.5 to 3.5 mpa , time of press moulding 5 min , temperature of press moulding from 130 to 190 c . selected results of measurements of physicochemical properties of coal - tar pitch and pitch - poly(ethylene terephthalate ) compositions are presented in table 1 . the addition of waste pet significantly influenced the softening point of ctp . with the increase of pet in the compositions , the softening point increased . for the composition containing 50 wt% of pet , the softening point compared to the unmodified pitch increased by 129 c . with the increase of waste pet in the compositions , the cooking value decreased and so the yield of residue after high - temperature carbonization process . in particular , significant changes occurred for compositions containing 50 wt% of the waste , where coking value was lower by 15 wt% compared to coal - tar pitch . the content of ti component in pitch - pet compositions was 4569 wt% and qi components was 1363 wt% . with the increase of waste in the compositions , the content of ti and qi components increased . in fig . 1 , microscopic images of pitch - pet compositions obtained using fluorescence microscope are presented . on obtained microscopic images , similarly as in the case of petroleum bitumens , carbon bitumen phase is observed in black colour.fig . 1microscopic images of the structure of pitch - pet compositions containing the following : a 10 wt% of waste pet , b 25 wt% of waste pet , and c 50 wt% of waste pet microscopic images of the structure of pitch - pet compositions containing the following : a 10 wt% of waste pet , b 25 wt% of waste pet , and c 50 wt% of waste pet structures of compositions containing 25 wt% pet , had uniform level of dispersion , and dispersed polymer particles had round and regular shapes . the increase of waste pet amount in compositions caused the increase of the amount and size of dispersed particles . for the composition containing 50 wt% of waste poly(ethylene terephthalate ) agglomerates of irregular shape could be observed , which were formed from aggregates containing polymer macromolecules and probably components of coal - tar pitch . it has to be presumed that with the increase of waste pet amount in compositions , the development of micellar areas occurred due to strong physical interactions between the components , which is indicated by i.a . , high softening temperatures . selected results of measurements of physicochemical properties of pitch - poly(methylene methacrylate ) compositions are presented in table 1 . addition of waste pmma influenced the change of physicochemical properties of coal - tar pitch . for the compositions containing 10 or 25 wt% of waste pmma , the softening point compared to the unmodified pitch increased by 30 and 38 c , respectively . the highest softening point was observed for the compositions containing 50 wt% of waste pmma . addition of waste pmma into ctp caused the decrease of coking value and so the yield of residue after high - temperature carbonization process . an exemption from this rule was the composition containing 10 wt% of waste pmma , which had higher coking value than ctp ( by 7.05 wt% ) . in particular , significant changes occurred for compositions containing 50 wt% of the waste , where coking value was lower by 24.20 wt% compared to coal - tar pitch . increase of waste pmma addition into ctp caused the decrease of content of ti components and increase of content of qi components . the pitch - pmma compositions had lower content of ti components and had higher content of qi components compared to coal - tar pitch . an exemption from this rule was the composition containing 10 wt% of waste pmma , which had higher content of ti components by 1.41 wt% compared to ctp . in fig . 2 , microscopic images of pitch - pmma compositions obtained using fluorescence microscope are presented.fig . 2microscopic images of the structure of pitch - pmma compositions containing the following : a 10 wt% of waste pmma , b 25 wt% of waste pmma , c 50 wt% of waste pmma microscopic images of the structure of pitch - pmma compositions containing the following : a 10 wt% of waste pmma , b 25 wt% of waste pmma , c 50 wt% of waste pmma structures of pitch - waste pmma compositions containing up to 25 wt% of additive assumed the shapes of round particles of different shapes , chaotically dispersed in continuous phase of the bitumen . with the increase of waste pmma in compositions increased the amount and size of dispersed aggregates of irregular shapes , composed of macromolecules of a polymer and probably of components of coal - tar pitch . for the composition containing 50 wt% of waste pmma , phase inversion was observed in the dispersion . selected results of measurements of physicochemical properties of pitch - phenol - formaldehyde resin compositions softening point increased with increasing content of waste pf . in the case of compositions containing 50 wt% of this component , the measurements of softening point was not possible because of the impossibility to melt them . for the compositions containing pf , independently from the amount of waste , coking values were similar to the value of coal - tar pitch . addition of waste pf into ctp caused changes of group composition of the bitumen . with the increase of waste pf in the compositions , the content of ti components increased . for the composition containing 50 wt% of waste pf , the content of ti components increased by 54.33 wt% compared to the unmodified pitch . the content of qi components in pitch - pf compositions was 4.24.5 wt% and was lower than ctp . an exemption from this rule was the composition containing 10 wt% of waste pf , which had higher content of qi components compared to coal - tar pitch ( by 3 wt% ) . in fig . 3 , structures of compositions containing 25 wt% of pf , similarly as in the case of compositions containing pet in the amount of 25 wt% , exhibited uniform level of dispersion , and dispersed particles had round shape . further increase of the amount of phenol - formaldehyde resin in compositions up to 50 wt% caused formation of agglomerates of irregular shapes , being assemblies of aggregates composed of polymer macromolecules and probably of components of coal - tar pitch ( fig . 3microscopic images of the structure of pitch - pf compositions containing the following : a 10 wt% of waste pf , b 25 wt% of waste pf , c 50 wt% of waste pf microscopic images of the structure of pitch - pf compositions containing the following : a 10 wt% of waste pf , b 25 wt% of waste pf , c 50 wt% of waste pf selected results of measurements of physicochemical properties of coal - tar pitch and pitch - poly(ethylene terephthalate ) compositions are presented in table 1 . the addition of waste pet significantly influenced the softening point of ctp . with the increase of pet in the compositions , the softening point increased . for the composition containing 50 wt% of pet , the softening point compared to the unmodified pitch increased by 129 c . with the increase of waste pet in the compositions , the cooking value decreased and so the yield of residue after high - temperature carbonization process . in particular , significant changes occurred for compositions containing 50 wt% of the waste , where coking value was lower by 15 wt% compared to coal - tar pitch . the content of ti component in pitch - pet compositions was 4569 wt% and qi components was 1363 wt% . with the increase of waste in the compositions , the content of ti and qi components increased . in fig . 1 , microscopic images of pitch - pet compositions obtained using fluorescence microscope are presented . on obtained microscopic images , similarly as in the case of petroleum bitumens , carbon bitumen phase is observed in black colour.fig . 1microscopic images of the structure of pitch - pet compositions containing the following : a 10 wt% of waste pet , b 25 wt% of waste pet , and c 50 wt% of waste pet microscopic images of the structure of pitch - pet compositions containing the following : a 10 wt% of waste pet , b 25 wt% of waste pet , and c 50 wt% of waste pet structures of compositions containing 25 wt% pet , had uniform level of dispersion , and dispersed polymer particles had round and regular shapes . the increase of waste pet amount in compositions caused the increase of the amount and size of dispersed particles . for the composition containing 50 wt% of waste poly(ethylene terephthalate ) agglomerates of irregular shape could be observed , which were formed from aggregates containing polymer macromolecules and probably components of coal - tar pitch . it has to be presumed that with the increase of waste pet amount in compositions , the development of micellar areas occurred due to strong physical interactions between the components , which is indicated by i.a . selected results of measurements of physicochemical properties of pitch - poly(methylene methacrylate ) compositions are presented in table 1 . addition of waste pmma influenced the change of physicochemical properties of coal - tar pitch . for the compositions containing 10 or 25 wt% of waste pmma , the softening point compared to the unmodified pitch increased by 30 and 38 c , respectively . the highest softening point was observed for the compositions containing 50 wt% of waste pmma . addition of waste pmma into ctp caused the decrease of coking value and so the yield of residue after high - temperature carbonization process . an exemption from this rule was the composition containing 10 wt% of waste pmma , which had higher coking value than ctp ( by 7.05 wt% ) . in particular , significant changes occurred for compositions containing 50 wt% of the waste , where coking value was lower by 24.20 wt% compared to coal - tar pitch . increase of waste pmma addition into ctp caused the decrease of content of ti components and increase of content of qi components . the pitch - pmma compositions had lower content of ti components and had higher content of qi components compared to coal - tar pitch . an exemption from this rule was the composition containing 10 wt% of waste pmma , which had higher content of ti components by 1.41 wt% compared to ctp . in fig . 2 , microscopic images of pitch - pmma compositions obtained using fluorescence microscope are presented.fig . 2microscopic images of the structure of pitch - pmma compositions containing the following : a 10 wt% of waste pmma , b 25 wt% of waste pmma , c 50 wt% of waste pmma microscopic images of the structure of pitch - pmma compositions containing the following : a 10 wt% of waste pmma , b 25 wt% of waste pmma , c 50 wt% of waste pmma structures of pitch - waste pmma compositions containing up to 25 wt% of additive assumed the shapes of round particles of different shapes , chaotically dispersed in continuous phase of the bitumen . with the increase of waste pmma in compositions increased the amount and size of dispersed aggregates of irregular shapes , composed of macromolecules of a polymer and probably of components of coal - tar pitch . for the composition containing 50 wt% of waste pmma , phase inversion was observed in the dispersion . selected results of measurements of physicochemical properties of pitch - phenol - formaldehyde resin compositions are presented in table 1 . softening point increased with increasing content of waste pf . in the case of compositions containing 50 wt% of this component , the measurements of softening point was not possible because of the impossibility to melt them . for the compositions containing pf , independently from the amount of waste , coking values were similar to the value of coal - tar pitch . addition of waste pf into ctp caused changes of group composition of the bitumen . with the increase of waste pf in the compositions , the content of ti components increased . for the composition containing 50 wt% of waste pf , the content of ti components increased by 54.33 wt% compared to the unmodified pitch . the content of qi components in pitch - pf compositions was 4.24.5 wt% and was lower than ctp . an exemption from this rule was the composition containing 10 wt% of waste pf , which had higher content of qi components compared to coal - tar pitch ( by 3 wt% ) . in fig . 3 , structures of compositions containing 25 wt% of pf , similarly as in the case of compositions containing pet in the amount of 25 wt% , exhibited uniform level of dispersion , and dispersed particles had round shape . further increase of the amount of phenol - formaldehyde resin in compositions up to 50 wt% caused formation of agglomerates of irregular shapes , being assemblies of aggregates composed of polymer macromolecules and probably of components of coal - tar pitch ( fig . 3microscopic images of the structure of pitch - pf compositions containing the following : a 10 wt% of waste pf , b 25 wt% of waste pf , c 50 wt% of waste pf microscopic images of the structure of pitch - pf compositions containing the following : a 10 wt% of waste pf , b 25 wt% of waste pf , c 50 wt% of waste pf the application of fluorescence microscope for the evaluation of colloidal structure of pitch - polymer compositions can be a valuable supplement to other methods used to evaluate the homogenicity and level of dispersion of a polymer in bitumen - polymer compositions , and also it can allow to explain the phenomena occurring during processes of modification of coal - originated bitumens with macromolecular compounds . the changes of physicochemical properties of coal - tar pitch depended on the type and amount of applied waste polymer . addition of waste pet into ctp caused increase of softening point and the amount of components insoluble in toluene and quinoline , while coking value in pitch - pet compositions decreased . addition of waste pmma into ctp caused increase softening point and the amount of components insoluble in quinoline , decrease coking value and the amount of components insoluble in toluene . addition of waste pf into ctp caused increase softening point and the amount of components insoluble in toluene , decrease the amount of components insoluble in quinoline and caused slightly changes of coking value . pitch - polymer compositions can be treated as microheterogeneous systems , colloidal and biphase , generally exhibiting uniform dispersion of particles composed of polymer macromolecules and probably of components of coal - tar pitch . the size of dispersed particles depends on the type and amount of waste polymer added to the coal - tar pitch . observed microscopic images allowed to point out the following dependences between the properties of pitch - polymer compositions and their structure : for pitch - pet compositions , the increase of the size of dispersed aggregates caused the increase of softening point and content of ti and qi components and reduction of coking value , for compositions containing waste pmma , the increase of the size of dispersed aggregates caused the increase of softening point and content of qi components , reduction of coking value and content of ti components andfor pitch - pf compositions , the increase of the size of dispersed aggregates caused the increase of softening point and content of ti components and decrease content of qi components , while coking value changed insignificantly . for pitch - pet compositions , the increase of the size of dispersed aggregates caused the increase of softening point and content of ti and qi components and reduction of coking value , for compositions containing waste pmma , the increase of the size of dispersed aggregates caused the increase of softening point and content of qi components , reduction of coking value and content of ti components and for pitch - pf compositions , the increase of the size of dispersed aggregates caused the increase of softening point and content of ti components and decrease content of qi components , while coking value changed insignificantly . current techniques in acl surgery have been associated with satisfactory long - term results in the majority of patients . however , there remains a considerable subset , up to 30% of patients , with unsatisfactory outcomes [ 2 , 7 ] . specifically , patients report problems relating to rotational instability and return to previous level of activity [ 3 , 10 ] . it has been suggested that a more anatomical approach to restore the original acl anatomy may benefit these patients [ 4 , 17 ] . some authors have advocated placing a single graft in a position closer to the oblique femoral attachment of the acl [ 14 , 15 ] . however , it is not possible to fully restore normal knee kinematics with a single graft , regardless of the position [ 13 , 16 ] . the double bundle reconstruction technique ( dbt ) for acl reconstruction aims at restoring the acl anatomy with its two bundles and is gaining popularity [ 11 , 17 ] . anatomic studies have demonstrated the presence of two functional bundles within the acl , the anteromedial ( am ) and the posterolateral ( pl ) bundle [ 1 , 8 ] . although it is somewhat of a simplification , the double bundle description of the acl is generally accepted as an anatomic model for understanding the complex structure and function of the ligament [ 6 , 9 ] . the am bundle often obscures the pl bundle , and it may appear that only one bundle is present without careful inspection . the goal of this study was to describe the presence of the double bundle structure from an arthroscopic point of view , and to evaluate the value of different portals in knee arthroscopy . we prospectively examined 60 knees during standard arthroscopy . in each knee , the double bundle acl structure was evaluated , along with the usefulnes of different portals for visualization . all knees that were included in the study had a previous x - ray and mri in order to rule out any significant changes to the bone , and to ensure that the acl was intact . all patients were less than 60 years and had no history of acl injury . surgical indications for the 60 total subjects examined included 31 cases treated for meniscal findings alone , 21 treated for articular cartilage findings alone , and 8 cases of a combined repair of meniscus and cartilage . there were 25 female and 35 male knees , 29 right knees , and 31 left knees included in the study . age distribution ranged between 16 and 60 , with an average age of 40.2 years . arthroscopy started with an anterolateral portal ( alp ) located just lateral to the patellar tendon using the inferior pole of the patella as a vertical landmark , and an anteromedial portal ( amp ) approximately 0.5 cm medial to the edge of the patella tendon , 1 cm superior to the joint line , and 1 cm inferior to the tip of the patella . for each knee , the acl anatomy and the visibility of the pl bundle through the alp and amp were evaluated with and without retraction of the am bundle according to the description of the two bundles by girgis et al . and arnoczky . gross biomechanics of the two bundles using a probe were also assessed . for statistical analysis with the arthroscope in the alp , we were able to distinguish the am and pl bundle in 17 cases ( 28.3% ) ( fig . 1 ) . in the remaining cases ( n = 43 , 71.7% ) , the pl bundle was obscured by the am bundle , and visualization was only possible with retraction of the am bundle with a probe ( fig . 2 ) . switching the arthroscope to the amp , differentiation of the am and pl bundle without using a probe to retract am was possible in 40 cases ( 66.7% ) ( fig . 3 ) . in the remaining cases ( n = 20 , 33.3% ) , visualization of the pl bundle was possible only after retraction of the am bundle ( table 1 ) . there was a statistically significant better visibility of the pl bundle using the amp ( p < 0.05).fig . 2anterolateral portal : pl bundle with retraction of am bundle , pl bundle loose with knee flexionfig . bundlepl bundleanterolateral portaln = 17 ( 28.3%)anteromedial portaln = 40 ( 66.7% ) * * p < 0.05 anterolateral portal : pl bundle without retraction of am bundle anterolateral portal : pl bundle with retraction of am bundle , pl bundle loose with knee flexion anteromedial portal : pl bundle without retraction of am bundle visualization of the pl bundle the femoral insertion site of the acl was semilunar at the inner surface of the lateral condyle . the centre of the pl bundle visualized more shallow than the centre of the am bundle with the knee held in 90 flexion , while both insertion sites were oriented horizontally ( fig . 4 ) . the pl bundle insertion was located at the posterolateral aspect of the tibial attachment with a close approximation of the pl bundle to the posterior root of the lateral meniscus . gross assessment using a probe while applying a flexion - extension motion pattern to the knee demonstrated a taut am bundle throughout the range of motion and a tightening of the pl bundle with knee - extension ( figs . 2 , 5 ) . pl bundle tight with knee extension anteromedial portal : pl bundle without retraction of am bundle anteromedial portal : pl bundle tight with knee extension the principal finding of the present study was that it is possible to distinguish the double bundle structure in acl anatomy as described by girgis et al . and arnoczky by applying a two portal knee arthroscopy technique . the visualization of the acl anatomy seems to be improved through the use of a medial portal . the anteromedial portal not only helps in visualization of the double bundle structure but also in distinguishing the am and pl portions of the femoral attachment sites . consistent with the literature , we found the pl bundle tightening when the knee is extended [ 6 , 12 ] . this study is limited by the fact that we observed only acl intact knees and did not consider cases with a torn acl , where the anatomic position of the attachment sites is sometimes obscured by the knee injury and bony changes that occur between injury and reconstruction . therefore , we stress the need for further arthroscopic evaluation of different portals in acl reconstruction . the clinical relevance of this study is that it can be beneficial to establish an additional medial working portal . the amp can be used for the arthroscope and a better visualization of the femoral insertion site of the acl at the inner surface of the lateral condyle and a precise femoral tunnel placement can be achieved . reported the use of an additional medial portal without any increase in morbidity to patients for acl surgery .

in this work , the results of studies on the evaluation of colloidal structure of coal - tar pitch compositions with selected waste polymers by fluorescence microscope . for pitch - polymer compositions containing 1050 wt% waste polymer , softening point , coking value and content of components insoluble in toluene and quinoline were carried out . the results indicate that pitch - polymer compositions can be treated as microheterogeneous systems , colloidal and biphase , generally exhibiting uniform dispersion of particles composed of polymer macromolecules and probably of components of coal - tar pitch . in order to describe the arthroscopic presence of the double bundle structure and to evaluate the value of different portals in knee arthroscopy , we assessed the am and pl bundle anatomy . we prospectively examined the knees of 60 patients undergoing arthroscopic surgery for pathology unrelated to the acl . arthroscopy was performed in a two portal technique using an anterolateral ( alp ) and an anteromedial ( amp ) portal . with the arthroscope in the alp , we could distinguish an am and pl bundle in 28% . switching the arthroscope to the amp , differentiation of the bundles was possible in 67% . in all remaining cases visualization of the pl bundle was possible after retraction of the am bundle . use of amp increased visualization of the pl bundle . it seems reasonable to perform arthroscopy for acl reconstruction with the arthroscope in the amp and to establish an additional medial working portal to increase the visualization of the femoral acl insertion sites for optimal femoral tunnel placement .

many stroke patients with stroke have a balance problems while standing because they tend to put their weight on their non - paralyzed lower limb to increase swing of their upper body1 . falling accidents due to such decline in balance ability is on the increase annually . even when there is no injury from falling , frequent falling can sufficiently disrupt physical and social activities2 . because deterioration of muscular strength and balance ability increases the risk of falling , it is significantly important to perform exercises to promote upper limb muscular strength and training for balance3 . regular exercise of walking and for muscular strength and balance may increase muscular strength and balance to prevent falling of patients4 . walking , in particular , is known to be a safe type of aerobic exercise for patients , having the merit that it can be performed gradually by considering appropriate intensity , frequency , period , and phases of exercise for abilities of each patient5 . recently efficiency of walking training using a treadmill has attracted more attention as when such training has been reported to have better effects than walking on even ground . some researchers reported that walking on a treadmill is effective in improving balance and walking ability6 , 7 . with recent developments of relevant computer programs , new methods of rehabilitation exercise such exercises can increase interest and participation of patients in treatment and improve their significant functions by enabling them to perform various forms of tasks appropriate for goals of individual patients in virtual reality . virtual reality indicates a type of interactive simulation using computer hardware and software , in which users can have close - to - reality experiences8 . virtual reality training using visual feedback enables the participants to undergo the training while seeing their movements with their own eyes , a feature that helps them adjust their inaccurate body center caused by body image damage suffered by most stroke patients . in addition , training serves as a catalyst in active participation and performance of tasks by inducing interest and pleasure , providing immediately visual feedback on the result to enhance motor education ability9 . many recent researches of virtual reality games with higher accessibility reported that such games showed significant effects on voluntary control and coordination of children with cerebral palsy and increases in the motor ability of patients with parkinson disease10 . those types of games were also effective on recovery of upper limb functions and induced significant improvement in balance and walking of stroke patients , showing that the games can be used as an effective method of intervention11 , 12 . as a means of overcoming limitations of the existing intervention methods , virtual reality shows an availability it has to being applied in the rehabilitation of stroke patients . however , devices of virtual reality in previous studies are so large that they are difficult to be used in general social institutes or homes in terms of scale and cost . furthermore , they are targeted to a small number of patients . in this context , the purpose of this study was to perform treadmill exercise and virtual reality training exercise using nintendo wii , a device that can be easily used without regard to place or facility , in order to investigate the effects on balance and walking of stroke patients . all of the subjects were sufficiently explained of this study and voluntarily consented to their participation in the experiment . this study was approved by the research agency , and all participants provided written informed consent . those who could perform communication , comply with instructions in this study , perform balancing and walking independently , had no pain limiting execution of exercise , and had no disability in sight , hearing , and the vestibular organs were selected to be the subjects of this study . the forty stroke patients were randomly divided into two exercise program groups with one group using virtual reality training ( n=20 ) and another group using a walking exercise program using a treadmill ( n=20 ) . age for the virtual reality group , the age was 62.2 7.2 years old , the height was 162.8 6.7 cm , the weight was 58.1 5.4 kg , and the onset period was 30.4 5.4 months . age for the treadmill group , the age was 63.2 5.4 years old , the height was 164.7 7.5 cm , the weight was 60.2 4.5 kg , and the onset period was 31.6 7.4 months . the virtual reality group underwent training using the wii - fit software by nintendo , japan . when playing the nintento wii , the participants used wireless controllers to interact with the avatars on the screen via the virtual reality motion detection system . the controllers were attached by acceleration detecting sensors responding to changes in direction and speed . due to the detector being installed on the television , the screen showed the movements of the controller just as the player performed the movements13 . the virtual reality system used in this study was the wii board balance system , in which users played games while the balancing board sensed weight shift and distribution . the subjects of the virtual reality group performed yoga , muscular strength exercise , aerobic exercise , and balancing exercise , all for 10 minutes each for a total of 40 minutes . the treadmill group used low - speed treadmills for patients to directly control their speed based on their walking ability while walking for 40 minutes . the treadmills were equipped with handles on the front and on both sides , which the users could grip when they lost balance walking . we used a pedoscan ( rsscan 1 m , germany ) to measure balance of the subjects . the device consisted of 4,096 sensors for measuring pressures on both feet in static standing , computerizing left / right weight - bearing and anterior / posterior weight - bearing by separating each area . the patients were asked to stand up straight on the device without any movement for 30 seconds . the device was attached to the dominant foot of a subject , measuring the anterior and posterior pressures of the foot by the pressure detecting sensors on its insole . the insole between the foot and the board sensed the vertical reaction force to measure the stance phase , swing phase , cadence of the dominant foot . the measured signals were temporarily saved by the portable compact controller attached to the ankle by receiving the walking data of the subject and then were computerized and calculated . before the walking exercise , the smart step insoles were inserted in the shoes and were injected with air to maintain appropriate pressure . after putting on the shoes and being attached to the controller on their ankles , the subjects walked along a straight footpath for 10 meters , looking straight ahead . a paired t - test was used for before and after comparisons , and an independent t - test was used for a between group comparison with a significance level of =0.05 . for balance , there were significant differences in both the virtual reality group and the treadmill group after completing of the exercise program . for walking , the virtual reality group showed significant differences after completing of the exercise program . significant differences between the groups were shown in balance after completing of the exercise program ( table 1table 1.the comparison of balance and gait in virtual reality and treadmillperiodvirtual reality group ( n=20 ) treadmill group ( n=20)m sdm sdleft / right weight - bearing ( % ) pre17.1 5.516.9 6.5post10.6 4.8**13.1 5.8*anterior / posterior weight - bearing ( % ) pre15.4 6.715.7 4.6post10.3 4.7**12.9 5.1*affected side stance phase ( % ) pre53.7 6.454.2 7.6post57.5 5.7 * 55.3 6.4affected side swing phase ( % ) pre46.2 6.445.7 7.6post42.4 5.7 * 44.6 6.4cadence ( steps / min)pre44.2 5.443.7 6.4post48.1 6.1 * 45.2 6.1*paired t - test , independent t - test , * p<0.05 , * * p<0.01 ) . * paired t - test , independent t - test , * p<0.05 , * * p<0.01 falling can occur in all age groups , but older people and patients show higher frequency of falling . planned and consistent exercise is needed to prevent falling and strengthen muscles , and exercises promoting weight bearing , resistance , and aerobic process are known to be effective for such purposes14 . exercises for lower limb muscular strength and balancing exercises are effective for the prevention of falling and improvement of stamina of patients , but such exercises have some demerits because the content is simple and thus boring . to supplement the demerits , new methods of exercise owing to development of scientific techniques have been recently introduced where patients can perform various tasks in virtual reality15 , 16 . therapeutic intervention via virtual reality is more advantageous than other intervention methods in that it can provide relatively easy environmental control , availability in selecting environments , training based on patients abilities via phase - in task difficulty , rapid and precise feedback on performing tasks , and self - learning in safe environment17,18,19 . we applied an eight - week exercise program to stroke patients who were divided into a virtual reality exercise group and a treadmill walking group , in order to investigate effects of each exercise on balance and walking . the results of measuring balance and walking effects as the purpose of the virtual reality exercise program showed that significant differences were shown in all the domains of the left / right and anterior / posterior weight - bearing in balancing and the stance phase , the swing phase , and the cadence in walking . for the treadmill group , significant differences were displayed in the left / right and anterior / posterior weight - bearing in balancing . although the walking exercise program using a treadmill showed significant differences in balancing ability , the exercise program using virtual reality training displayed significant differences in balancing and walking because the patients could directly adjust their weight center and shift due to visual feedback . in addition , various similar programs such as jogging may further influence their balancing and walking . the results of this study suggest that virtual reality training may be appropriate to provide planned and consistent exercises for stroke patients who need improved balance and walking ability by giving visual feedback to induce their interest . meanwhile , the results of this study are consistent with those reporting increases in left / right , anterior / posterior stability of stroke patients who performed of virtual reality training , or reporting significant improvement in the berg balance test for the elderly who underwent a training using virtual reality games20 . in addition , there were researches in which significant differences were observed in balancing ability of the elderly with their eyes open after they underwent a virtual reality exercise program , or in which significant differences were found in balancing ability of the elderly after an eight - week virtual reality exercise program . meanwhile , the increase in walking ability shown in this study was consistent with the results of studies in which electrotherapy based on virtual reality significantly increased walking speed of stroke patients , or in which exercise using visual feedback was effective on the walking ability of stroke patients8 , 14 , 21 . in this study also , virtual reality training was effective in producing positive improvements in balance and walking of stroke patients . however , there remains insufficient research related to virtual reality because the technology has not been fully commercialized yet . further studies are needed to deal with virtual reality for many patients to use it in performing various indoor exercise programs and developing their abilities . the results of this study may indicate that virtual reality training providing visual feedback enables stroke patients to directly adjust their wrong weight center and shift visually , and that it is appropriate for the patients who need improved balance and walking ability by inducing their interest for them to perform planned exercise on a consistent basis . since the introduction of antiretroviral therapy in 1996 , the clinical management of hiv infection revolves around a complex assemblage of potent pharmacological molecules that inhibit viral replication . the goal of this lifelong treatment regimen is to achieve ( and maintain ) viral suppression and to reach a threshold where copies of the virus are no longer detectable in the blood the lowest level of detection generally considered 4075 copies / ml ( hhs 2011 ) . an undetectable plasma viral load is widely interpreted as the marker of individual therapeutic success and is also used extensively to conceptualize health in the context of hiv ( persson et al . the person living with hiv who is able to achieve this kind of viral suppression through strict adherence can be understood as a good patient and deserving of the ongoing investment in their health , irrespective of the complexity of their lives and the numerous barriers to adherence many individuals face ( remien and mellins 2007 ) . however , in recent years , plasma viral load testing has become more than just a clinical tool to monitor treatment response at the individual level . increasingly , individual plasma viral load testing is being reported to public health agencies and is used to inform epidemiological surveillance and monitor the presence of the virus collectively ( terzian et al . in particular need of critical appraisal is the development of techniques to measure , map and monitor community viral load. this new epidemiological technology blurs the traditional boundaries of clinical care and public health work , thus raising questions about its implications . for the purpose of this article , we argue that community viral load should be examined in relation to the theoretical framework of foucault ( 1984 ) coined this concept to account for the ways through which capillary forms of power insert themselves into actions , attitudes , and discourses to produce a particular kind of person and population - productive , but docile and easily governed . miller and rose ( 2008 , p. 63 ) describe governmentality as a domain of strategies , techniques and procedures through which different forces seek to render programmes operable , and by means of which a multitude of connections are established between the aspirations of authorities and the activities of individuals and groups. these tactics , in their programmatic form , act directly and indirectly on the population ( and individuals who compose it ) to improve its conditions , welfare , wealth , longevity , health and so on ( foucault 1991 ) . these tactics become inseparable from a knowledge and understanding of processes related to population ( foucault 1991 ) . this form of governance is particularly concerned with processes that modulate health collectively rather than individually ; and this is why these processes have to be understood and managed using a broad range of tactics ( foucault 1991 ) . to govern , therefore , implies an ideological shift from the isolated person to the existing relations that are known to interfere with the production of a healthy population : relations between people and people , people and things , people and events , [ people and spaces] ( rose et al . these relations can then be administered directly or indirectly through tactics that make possible the optimization of health and so forth ( foucault 1991 ) . based on the work of foucault ( 1991 , 2003 ) , this article critically examines the use of community viral load as a new strategy of governmentality . also drawing on the work of miller and rose ( 2008 ) , this article further reconceptualizes specifically , it explores the deployment of community through the re - configuration of space , the problematization of viral concentrations in specific micro - locales , and the government ( in the foucauldian sense ) of specific bodies which are seen as it also examines community viral load as a necessary precondition forming the conditions of possibility for the recent shift to high impact prevention tactics that are being scaled up across north america . 2 ) describe community viral load as an aggregate biological measure of viral load for a particular geographic location for example the city of san francisco or a particular neighborhood and for a particular group of people who share socio - demographic characteristics. in order to determine the community viral load of any particular group or area of interest , one must take into account two distinct measures ; mean community viral load and total community viral load ( das et al . mean community viral load is the average of the most recent viral load of all reported hiv - positive persons in a particular target(ed ) population or geographical area ( das et al . total community viral load is the sum of the most recent viral loads of all reported hiv - positive persons in a particular target(ed ) population or geographical area ( das et al . these measurements make it possible to determine the mean average viral burden and the absolute level of the virus in any given population or geographical area . community viral load is a relatively new concept but has gained significant momentum in the field of hiv in recent years . this is due in part to the implementation of seek , test and treat ( now seek , test , treat and retain ) initiatives which rely on the premise that scaling up treatment collectively will result in a dramatic reduction of hiv transmission ( montaner et al . community viral load is considered by many researchers and clinicians to be a valuable marker of the direct relationship between hiv concentration ( in specific populations and geographical areas ) and overall hiv incidence ( wood et al . , community viral load has become an important concept under which to advocate for the expansion of testing and surveillance in the community and , most importantly , the reconceptualization of treatment as a prevention tool ( montaner et al . 2010 ) . recent studies in canada and the united states have demonstrated the value of measuring and mapping hiv viral load at the community level . in vancouver and across the province of british columbia ( canada ) , community viral load is increasingly being used as an epidemiological surveillance tool and an evaluation tool to monitor the outcomes of initiatives for the expansion of testing and treatment coverage ( montaner et al . 2010 ) . building on research findings , which suggest that community viral load is correlated with hiv incidence ( wood et al . 2009 ) , these initiatives were implemented across the province ( and more aggressively in the downtown eastside of vancouver ) and have led to a progressive decline in community viral load ( montaner et al . in some postal code areas of the province , however , the proportions of potentially infectious individuals ( as stated by the authors ) remain high regardless of the expansion of testing and treatment coverage ( lourenco et al . 2012 ) . similar findings have been reported in san francisco ( california , united states ) following the intensification of surveillance , the expansion of testing and earlier initiation of treatment ( das et al . 2011 ) . in addition to a documented decline in community viral load and new reported cases of hiv , the department of public health was also able to map areas with the highest concentration of viral load and further target them to produce improved outcomes ( das et al . areas such as tenderloin , south market , mission and castro were subsequently identified as hotspots ( as stated by the authors ) or areas with particularly high risk of hiv transmission due to the distribution of community viral load ( das et al . it was , therefore , recommended that more community - level interventions be implemented in these areas and that more aggressive prevention efforts ( including the use of treatment as a prevention tool ) be deployed to reduce community viral load . a similar mapping exercise was conducted in washington ( district of columbia , united states ) over a period of 4 years ( castel et al . ( 2012 ) included the most recent viral load data in the city , two indicators of socioeconomic status ( income and education ) , and race . their data were statistically analyzed and presented on various maps of the city of washington . these maps showed that the neighbourhoods with the worst socioeconomic indicators also had the highest hiv / aids prevalence rates ( castel et al . ( 2012 ) , can inform surveillance and the implementation of interventions that target populations with the highest viral load burden . it was determined that community viral load and its geospatial distribution are particularly useful to inform targeted public health interventions ( castel et al . similar arguments are presented by researchers who conducted studies on community viral load in new york city ( new york , united states ) from 2005 to 2009 ( laraque et al . based on their findings , they suggest that community viral load is valuable to public health agencies because it has the potential to identify high risk groups and target interventions to the groups whose viral control if achieved will most likely result in a rapid lowering of community viral load ( terzian et al . furthermore , it has the potential to identify groups at risk for sustained high viral load such as bronx residents who , according to terzian et al . ( 2012 ) , are more likely to have a detectable viral load and suffer from hiv - related disparities . identifying groups who are at risk or known to have sustained high viral load can thus provide an opportunity for the implementation of outreach initiatives in the community and the scale - up of efforts to educate , test , treat and engage members of the community in hiv care ( terzian et al . the inner workings of community viral load have not been critically examined in the hiv literature . the aim of this article is to interrogate the use of community viral load through a deductive process using theory to analyse empirics . theory makes it possible to engage in a critical analysis of a relatively new tool in the field of hiv and a limited body of empirical literature . it also allows us to disrupt normalizing discourses in the field of hiv and challenge the ways in which community viral load has been introduced as seemingly benign . this is particularly important considering that community viral load is now becoming normalized , standardized , and deployed in various public health agencies , without its assumptions and claims having been interrogated . in our view , it is imperative that researchers engage in discussions and debates around the use of community viral load and how it is intrinsically linked to the logics of governmentality . for this reason , we argue that a theory - based analysis of community viral load is of great value to these discussions and debates . community viral load links individual biomarkers and the concentration of the virus collectively through the production of particular kinds of spaces . these spaces are calculated , reconfigured , and imagined , with new levels of sophistication . first , community viral load signals an important shift in the way viral load is seen , imagined and represented both visually and spatially . specifically , we draw upon philo 's ( 2000 ) reading of foucault to expose three distinctive but interconnected representations of viral load . the first representation has to do with the tabulation of laboratory results that can attest to the progression of hiv and the response to treatment at the individual level . it draws a picture of how viral replication actually appears at every stage of hiv infection and what response is to be expected once treatment is initiated . here , tables and graphics of viral load patterns provide a reference point to assess individuals in the clinical setting and systematically position each of them accordingly in relation to an ideal patient with optimal viral suppression . it tracks viral replication within the body and penetrates its deepest recesses to expose the amount of copies of the virus in the blood , the breast milk , the sperm , the genital tract , the brain , the lymph nodes and so on . viral load is no longer seen as a laboratory value but rather as an evidence of viral activity in the body and a measure of infectiousness at the individual level . finally , the third representation allows for hiv to be located in space through mapping techniques . the location of the virus in space is compatible with foucault 's analysis of the shift from leper colony to plague city , wherein the risk of transmission is already present and requires the segmentation of space ( foucault 1990 ) . foucault explicitly linked mapping and government , and the need to have a rational plan to manage space ( crampton 2007 , p. 224 ) . in fact , he identified the ordering of space as central to the strategy for containment of biological risks and the government of processes that modulate health collectively rather than individually ( foucault 1990 , legg 2005 ) . these particular kinds of spaces take the form of specific neighbourhoods and locations where hiv is highly concentrated often located in geographical areas marked by high levels of marginalization , poverty , oppression and social exclusion . what is important here is not just that these spaces are rendered visible through viral load mapping and thus governable ( brown and knopp 2006 , 2010 ) , but the particular ways in which they are constructed based on the average viral burden of populations who occupy them and the absolute level of the virus that circulates among them . huxley ( 2006 ) explains that in order to better appreciate the articulation of spatial rationalities in the fabrication of governable spaces , we must pay close attention to the way authorities imagine these spaces and make them amenable to regulation . expanding on foucault 's work , huxley ( 2006 ) identifies three spatial rationalities : dispositional , generative and vitalist . for the purpose of this article , we will only refer to the dispositional and generative spatial rationalities . a dispositional rationality has to do with the production of boundaries and the spatial disposition of bodies ( huxley 2006 ) . it operates through the logics of quadrillage ( a term coined by foucault ) to prevent the spread of diseases and ensure a more effective regulation of processes that are responsible for epidemics ( huxley 2006 ) . in other words , it allows for the segmentation of space and the management of bodies to achieve a given end . entails systems or surveillance , as these are seen as essential to enforcing the ordering of spaces and bodies. generative rationality , on the other hand , concerns the effects of space on contagion , health , disease and death ( huxley 2006 ) . here , the problem is not so much the disposition of spaces but the concentration of diseased bodies in geographical areas ( huxley 2006 ) . these areas are seen in this imagination as particularly vulnerable and in need of attention ( brown and knopp 2010 , p. 394 ) . with this in mind , biomarkers become an important tool to circumscribe infectiousness and transmissibility ( or the risk thereof ) geographically . the emphasis is no longer on the disposition of diseased bodies in space but rather on areas where disease is highly concentrated and where the risk of transmission is intensified ( huxley 2006 ) . based on the work of huxley ( 2006 ) , we contend that the production of these spaces follows both dispositional and generational rationalities . by this , we mean that community viral load aims at drawing boundaries and mapping areas where the virus is unsuppressed areas that are then identified as vulnerable and in need of attention . we make a point here of referring to the virus because it is the real focus of this geographical ( and political ) undertaking even though we acknowledge that it reduces the bodies of people living with hiv to vectors of disease ( brown 1995 ) . community viral load , in fact , draws a rather partial and incomplete portrait of the hiv epidemic and continues to ignore the effect of context on hiv vulnerabilities -how the virus moves within a population and how it circulates across specific networks ( brown 1995 ) . what it does , however , is provide the necessary arguments to intensify surveillance , testing and prevention efforts in areas where we can find a number of people living with hiv whose viral load is unsuppressed. while our objective is not to portray people living with hiv as vectors of disease , we do have to stay true to the imaginations and rationales of those who see in community viral load a promising way to circumscribe infectiousness and transmissibility geographically . with this in mind , it is important to recognize that community viral load is increasingly being used as a tool to signal the existence of particular kinds of spaces , locate these spaces geographically , and allows for public health authorities to become more knowledgeable about the populations who occupy them . this knowledge generates new possibilities for the government of certain bodies which are seen as risky , dangerous , and in need of attention ( lupton 1995 ) . not surprisingly , it has led to the deployment of more aggressive interventions to lower the collective risk of acquiring or transmitting hiv , with little further consideration of the realities of people living with hiv on the ground , i.e. routine testing in institutions located in targeted areas , point of care testing in certain neighbourhoods , treatment as prevention initiatives in specific locations , and so forth . we shall now examine how community viral load cultivates specific ties between persons and communities through these new programmes of interventions . expanding on the work of michel foucault , miller and rose ( 2008 ) explain that the birth of the community marks a departure from governing a supposed collective social body to defining a new territory for the administration of collective life . this new territory has a number of significant features ( miller and rose 2008 ) . first , it calls on a particular re - configuration of space from a single , collective space to discrete communities which can be located geographically or constructed virtually ( i.e. lifestyle communities , moral communities , risk communities and so on ; miller and rose 2008 ) . second , it operates through the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 , p. 90 ) . in other words , it makes use of allegiances and specific ties between individuals and communities to regulate , reform and mobilize ( miller and rose 2008 ) . from this perspective , members of particular communities are encouraged to practice active personal responsibility and conduct themselves accordingly ( miller and rose 2008 ) . as a sign of social citizenship , they must take responsibility , they must show themselves capable of calculated action and choice , they must shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . third , it concerns the identification of individuals as members of particular communities and the work required to make these individuals aware of their allegiances , for example , with the disability community , the gay community or the aids community . here , the sense of community is created and promoted through the work of educators , campaigns , activists , manipulators of symbols , narratives and identifications ( miller and rose 2008 , p. 92 ) . that is to say that these particular communities are created and marketed despite the fact that they may appear to be natural and may resonate with our own personal identity ( miller and rose 2008 ) . drawing on a number of examples , miller and rose ( 2008 ) explain that government through community works , even when it works upon pre - existing bonds of allegiance , transforms them , invests them with new values , affiliates them to expertise and reconfigures relations ( p. 93 ) in a productive way . miller and rose ( 2008 ) explain that the construction of community involves various strategies for making individuals aware of their personal allegiances . one of these strategies is to raise awareness ( i.e. awareness campaigns ) , educate ( i.e. community training sessions ) , communicate ( i.e. media and other communication tools ) , and make sure that individuals identify themselves as members of that community ( miller and rose 2008 ) . drawing on health promotion programmes in hiv and aids , miller and rose ( 2008 ) explain that government through community produces new personal allegiances and works upon pre - existing ones to make individuals aware of their collective affiliations . these affiliations , explain miller and rose ( 2008 ) , are to be celebrated , encouraged , nurtured , shaped and instrumentalized in the hope of producing consequences that are desirable for all and for each ( p. 93 ) . affiliations to particular communities , then , create new relations of identification and , incidentally , new relations of mutual obligation ( miller and rose 2008 , p. 88 ) . as such , they open up questions of personal responsibilities and obligations . these questions are particularly relevant to our analysis because community viral load is not just a means of identification and community affiliation . it is also used to govern individuals who are located here and not there , that can be expected to behave and engage in certain ways and not others , and whose distribution is spatially correlated with other characteristics that makes them part of risk communities ( viral load , serological status , gender , ethnicity , socio - economic status , sexuality , etc . ) ( brown and knopp 2010 ) . from this perspective , members of risk communities must practice active personal responsibility and are expected to shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . those who do not exercise caution and live according to this moral code , however , are generally found in the margins of communities and their particular difficulties thus need to be addressed through the activities of various specialists each of whom is an expert in a particular problem ( miller and rose 2008 , p. 104 ) . these relations of expertise , explain miller and rose ( 2008 ) , operate at the level of the community . professional gaze , where expertise now focuses on conduct itself and the cognitive and moral organization of perception , intention , action and evaluation ( miller and rose 2008 , p. 106 ) . in these relations , the subject of expertise is conceived as an individual who lacks cognitive , emotional , practical , and ethical skills to take personal responsibility for rational self - management and fulfill his moral obligations ( miller and rose 2008 , p. 106 ) . this form of governance relies on the deployment of expert knowledge from various fields ( i.e. nursing , social work , medicine , etc . ) and practices of empowerment . empowerment , then , is a matter of experts teaching , coaxing , requiring their clients to conduct themselves within their respective communities , according to certain prescribed codes of active personal responsibility and moral obligations ( miller and rose 2008 , p. 106 ) . seek , test and treat initiatives which have recently been implemented in both canada and the united states , including community as a unit of identity , responsibilization and autonomization of people who reside in certain neighbourhoods , empowerment of individuals located in the margins , and expert management and outreach in risk communities. despite a seemingly emancipatory or rights - based agenda and the will to empower individuals who have been traditionally labelled as hard to reach ( patton 2011 ) , the deployment of these initiatives is indicative of the very form of government that miller and rose ( 2008 ) describe : the subject constituted through techniques of empowerment is being shaped into a better functioning and more responsible neoliberal actor empowered to make better decisions , able to fulfill moral obligations , and capable of committing to the health of his community . the experts are working directly in the community , relaying messages to individuals who have to be made aware of their personal allegiances to particular the community , as imagined through community viral load and the distribution of bodies in space , is now something to be programmed , researched and managed . the use of the term community is a powerful rhetorical tool and appeals to a common sense of belonging indeed , do we not all belong to some community ? yet , public health is not interested in all communities equally . as mentioned earlier , community viral load is part of a larger public health programme to promote high impact prevention using combinations of scientifically proven , cost - effective , and scalable interventions targeted to the right populations in the right geographic areas ( centers for disease control and prevention 2011 , p. 1 ) ( italics added for emphasis ) . within this new prevention framework , public health agencies are asked to identify communities where concentrations of the virus overlap with concentrations of substance users , gay and other men of have sex with men , transgender persons and racialized persons . community viral load provides a new tool for public health authorities to imagine particular communities ( legg 2005 ) in ways that are flexible , fluid , and can be adapted to different needs ( brown and knopp 2006 ) . this is inclusive of virtual communities of individuals who share particular risk factors but are geographically dispersed to communities of individuals who share geographical coordinates and whose virus is unsuppressed. what is important to understand here is not just that community viral load allows for these communities to be imagined , but that it is part of a large - scale operation to seek , test and treat more aggressively in target areas with the highest community viral load . this is part of the inner workings of community viral load ; it generates new knowledge , maps the presence of the virus collectively , makes room for a new form of surveillance , and in the end allows for the creation of specific targets , which are clustered around predominantly impoverished and marginalized neighbourhoods . seek , test and treat initiatives are arguably the best examples of high impact prevention efforts that specifically target such neighbourhoods and use on - site outreach to implement more aggressive forms of testing , surveillance and treatment -including mass testing fares with incentives meant to draw in the most marginalized . the rationale for these initiatives is largely based on the need to know community viral load and its geographic distribution . these initiatives appeal to the discourse of community ( lynn 2006 ) , with its shared culture , needs , and responsibilities , and relations of expertise ( miller and rose 2008 ) . we are particularly interested here in how community viral load creates or builds on existing personal allegiances and specific ties between individuals and communities to support the implementation of seek , test and treat initiatives ( miller and rose 2008 ) . not only is community viral load a potent tool to re - configure space ( as previously discussed ) and justify the need for public health to intervene more aggressively in certain neighbourhoods and locations , but it is also used as a device of identification . on the one hand , it concerns the identification of individuals who reside in areas where hiv is most heavily concentrated as members of a community. on the other hand , it reinforces the personal allegiances that these individuals may have to this community based on their serological status , their viral load , or the fact that they share a common fate as residents in neighbourhoods and locations where hiv is these individuals may , in turn , become actively involved in the deployment of community - level initiatives and engaged in outreach efforts to scale up hiv prevention . they may also become increasingly politicized . while this phenomenon may be beneficial for particular communities , it also tends to extend and reinforce the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 ) . seek , test and treat initiatives rely on the assumption that individuals who are made aware of their personal allegiances with a community will be more inclined to enrol , mobilize and act more responsibly ( miller and rose 2008 ) . one way to practice active personal responsibility is to take part in testing and link with prevention services . another way to practice active personal responsibility is to initiate antiretroviral treatment as soon as possible after diagnosis and demonstrate optimal treatment adherence . with this is mind , it is important to understand that seek , test and treat initiatives make room for new relations of expertise and new opportunities for experts to intervene directly in the community. in fact , these initiatives rely on a re - configuration of space and re - location of interventions traditionally done in clinical settings ; testing is now conducted in vans , shelters , community centres , gay bathhouses and drop - in services , results are communicated right away , and linkage to care is automatically provided , surveillance data are broadened and include geospatial analysis , individual viral load , census information and indicators of socioeconomic status , and treatment is initiated rapidly after diagnosis followed by ongoing follow - up and adherence counselling in the community . they also rely on the ongoing presence and visibility of experts in the community , working in neighbourhoods and locations that are most affected by the hiv / aids epidemic . again , this may be beneficial for some communities but it is important to acknowledge that the expansion of testing and treatment coverage are not substitute to the removal of vulnerabilities that place people at risk of infection in the first place ( which incidentally , overlap with vulnerabilities preventing access to [ hiv care and ] treatment ( nguyen et al . 2011 , p. 292 ) . while our goal is not refute the scientific evidence in support of high impact prevention , which is the guiding framework for seek , test and treat initiatives , we consider that these programmes require more reflection . in this article , we have acknowledged that this new framework is closely tied to the introduction of community viral and the need for more effective ways to govern bodies that are seen as contributing to the spread of hiv . in light of our analysis , we consider that this new framework has very little to do with the care of individuals who live with hiv/ aids and much more to do with the production of a healthy population and the control of certain bodies . it is concerned with matters of life and death , with health and illness , with infectiousness and transmissibility , and with the collective processes that contribute to the spread of hiv . it acts upon the health of the population as a whole by targeting geographical areas where hiv is highly concentrated , seeking and testing populations conceived as inherently risk - prone , vulnerable or unstable , and treating individuals who are found to be hiv positive in order to achieve individual viral suppression . therefore , in keeping with foucault 's ( 2003 ) work , we consider that community viral load has both individualizing and massifying effects . it was introduced to the field of hiv as a way to act upon communities who are located in specific geographical areas rather than individuals themselves . however , in order for it to be effective , it has to create specific ties between individuals who reside in areas where hiv is most heavily concentration and their respective risk communities. this has important implications for people living with hiv / aids , and may have broader implications for everyone who is being forced into these particular geographical areas these new ghettos that are marked by hiv and are under the radar of public health authorities . as such , we are concerned that community viral load has become a proxy for naming risk communities , but with new levels of sophistication . in particular , we are concerned with the potential to increase stigma directed at populations who occupy areas that are identified as highly virulent. this phenomenon has yet to be addressed in the literature and in research conducted on community viral load . community viral load links individual biomarkers and the concentration of the virus collectively through the production of particular kinds of spaces . these spaces are calculated , reconfigured , and imagined , with new levels of sophistication . first , community viral load signals an important shift in the way viral load is seen , imagined and represented both visually and spatially . specifically , we draw upon philo 's ( 2000 ) reading of foucault to expose three distinctive but interconnected representations of viral load . the first representation has to do with the tabulation of laboratory results that can attest to the progression of hiv and the response to treatment at the individual level . it draws a picture of how viral replication actually appears at every stage of hiv infection and what response is to be expected once treatment is initiated . here , tables and graphics of viral load patterns provide a reference point to assess individuals in the clinical setting and systematically position each of them accordingly in relation to an ideal patient with optimal viral suppression . it tracks viral replication within the body and penetrates its deepest recesses to expose the amount of copies of the virus in the blood , the breast milk , the sperm , the genital tract , the brain , the lymph nodes and so on . viral load is no longer seen as a laboratory value but rather as an evidence of viral activity in the body and a measure of infectiousness at the individual level . finally , the third representation allows for hiv to be located in space through mapping techniques . the location of the virus in space is compatible with foucault 's analysis of the shift from leper colony to plague city , wherein the risk of transmission is already present and requires the segmentation of space ( foucault 1990 ) . foucault explicitly linked mapping and government , and the need to have a rational plan to manage space ( crampton 2007 , p. 224 ) . in fact , he identified the ordering of space as central to the strategy for containment of biological risks and the government of processes that modulate health collectively rather than individually ( foucault 1990 , legg 2005 ) . these particular kinds of spaces take the form of specific neighbourhoods and locations where hiv is highly concentrated often located in geographical areas marked by high levels of marginalization , poverty , oppression and social exclusion . what is important here is not just that these spaces are rendered visible through viral load mapping and thus governable ( brown and knopp 2006 , 2010 ) , but the particular ways in which they are constructed based on the average viral burden of populations who occupy them and the absolute level of the virus that circulates among them . huxley ( 2006 ) explains that in order to better appreciate the articulation of spatial rationalities in the fabrication of governable spaces , we must pay close attention to the way authorities imagine these spaces and make them amenable to regulation . expanding on foucault 's work , huxley ( 2006 ) identifies three spatial rationalities : dispositional , generative and vitalist . for the purpose of this article a dispositional rationality has to do with the production of boundaries and the spatial disposition of bodies ( huxley 2006 ) . it operates through the logics of quadrillage ( a term coined by foucault ) to prevent the spread of diseases and ensure a more effective regulation of processes that are responsible for epidemics ( huxley 2006 ) . in other words , it allows for the segmentation of space and the management of bodies to achieve a given end . entails systems or surveillance , as these are seen as essential to enforcing the ordering of spaces and bodies. generative rationality , on the other hand , concerns the effects of space on contagion , health , disease and death ( huxley 2006 ) . here , the problem is not so much the disposition of spaces but the concentration of diseased bodies in geographical areas ( huxley 2006 ) . these areas are seen in this imagination as particularly vulnerable and in need of attention ( brown and knopp 2010 , p. 394 ) . with this in mind , biomarkers become an important tool to circumscribe infectiousness and transmissibility ( or the risk thereof ) geographically . the emphasis is no longer on the disposition of diseased bodies in space but rather on areas where disease is highly concentrated and where the risk of transmission is intensified ( huxley 2006 ) . based on the work of huxley ( 2006 ) , we contend that the production of these spaces follows both dispositional and generational rationalities . by this , we mean that community viral load aims at drawing boundaries and mapping areas where the virus is unsuppressed areas that are then identified as vulnerable and in need of attention . we make a point here of referring to the virus because it is the real focus of this geographical ( and political ) undertaking even though we acknowledge that it reduces the bodies of people living with hiv to vectors of disease ( brown 1995 ) . community viral load , in fact , draws a rather partial and incomplete portrait of the hiv epidemic and continues to ignore the effect of context on hiv vulnerabilities -how the virus moves within a population and how it circulates across specific networks ( brown 1995 ) . what it does , however , is provide the necessary arguments to intensify surveillance , testing and prevention efforts in areas where we can find a number of people living with hiv whose viral load is unsuppressed. while our objective is not to portray people living with hiv as vectors of disease , we do have to stay true to the imaginations and rationales of those who see in community viral load a promising way to circumscribe infectiousness and transmissibility geographically . with this in mind , it is important to recognize that community viral load is increasingly being used as a tool to signal the existence of particular kinds of spaces , locate these spaces geographically , and allows for public health authorities to become more knowledgeable about the populations who occupy them . this knowledge generates new possibilities for the government of certain bodies which are seen as not surprisingly , it has led to the deployment of more aggressive interventions to lower the collective risk of acquiring or transmitting hiv , with little further consideration of the realities of people living with hiv on the ground , i.e. routine testing in institutions located in targeted areas , point of care testing in certain neighbourhoods , treatment as prevention initiatives in specific locations , and so forth . we shall now examine how community viral load cultivates specific ties between persons and communities through these new programmes of interventions . expanding on the work of michel foucault , miller and rose ( 2008 ) explain that the birth of the community marks a departure from governing a supposed collective social body to defining a new territory for the administration of collective life . this new territory has a number of significant features ( miller and rose 2008 ) . first , it calls on a particular re - configuration of space from a single , collective space to discrete communities which can be located geographically or constructed virtually ( i.e. lifestyle communities , moral communities , risk communities and so on ; miller and rose 2008 ) . second , it operates through the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 , p. 90 ) . in other words , it makes use of allegiances and specific ties between individuals and communities to regulate , reform and mobilize ( miller and rose 2008 ) . from this perspective , members of particular communities are encouraged to practice active personal responsibility and conduct themselves accordingly ( miller and rose 2008 ) . as a sign of social citizenship , they must take responsibility , they must show themselves capable of calculated action and choice , they must shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . third , it concerns the identification of individuals as members of particular communities and the work required to make these individuals aware of their allegiances , for example , with the disability community , the gay community or the aids community . here , the sense of community is created and promoted through the work of educators , campaigns , activists , manipulators of symbols , narratives and identifications ( miller and rose 2008 , p. 92 ) . that is to say that these particular communities are created and marketed despite the fact that they may appear to be natural and may resonate with our own personal identity ( miller and rose 2008 ) . drawing on a number of examples , miller and rose ( 2008 ) explain that government through community works , even when it works upon pre - existing bonds of allegiance , transforms them , invests them with new values , affiliates them to expertise and reconfigures relations ( p. 93 ) in a productive way . miller and rose ( 2008 ) explain that the construction of community involves various strategies for making individuals aware of their personal allegiances . one of these strategies is to raise awareness ( i.e. awareness campaigns ) , educate ( i.e. community training sessions ) , communicate ( i.e. media and other communication tools ) , and make sure that individuals identify themselves as members of that community ( miller and rose 2008 ) . drawing on health promotion programmes in hiv and aids , miller and rose ( 2008 ) explain that government through community produces new personal allegiances and works upon pre - existing ones to make individuals aware of their collective affiliations . these affiliations , explain miller and rose ( 2008 ) , are to be celebrated , encouraged , nurtured , shaped and instrumentalized in the hope of producing consequences that are desirable for all and for each ( p. 93 ) . affiliations to particular communities , then , create new relations of identification and , incidentally , new relations of mutual obligation ( miller and rose 2008 , p. 88 ) . these questions are particularly relevant to our analysis because community viral load is not just a means of identification and community affiliation . it is also used to govern individuals who are located here and not there , that can be expected to behave and engage in certain ways and not others , and whose distribution is spatially correlated with other characteristics that makes them part of risk communities ( viral load , serological status , gender , ethnicity , socio - economic status , sexuality , etc . ) ( brown and knopp 2010 ) . from this perspective , members of risk communities must practice active personal responsibility and are expected to shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . those who do not exercise caution and live according to this moral code , however , are generally found in the margins of communities and their particular difficulties thus need to be addressed through the activities of various specialists each of whom is an expert in a particular problem ( miller and rose 2008 , p. 104 ) . these relations of expertise , explain miller and rose ( 2008 ) , operate at the level of the community . professional gaze , where expertise now focuses on conduct itself and the cognitive and moral organization of perception , intention , action and evaluation ( miller and rose 2008 , p. 106 ) . in these relations , the subject of expertise is conceived as an individual who lacks cognitive , emotional , practical , and ethical skills to take personal responsibility for rational self - management and fulfill his moral obligations ( miller and rose 2008 , p. 106 ) . this form of governance relies on the deployment of expert knowledge from various fields ( i.e. nursing , social work , medicine , etc . ) and practices of empowerment . empowerment , then , is a matter of experts teaching , coaxing , requiring their clients to conduct themselves within their respective communities , according to certain prescribed codes of active personal responsibility and moral obligations ( miller and rose 2008 , p. 106 ) . seek , test and treat initiatives which have recently been implemented in both canada and the united states , including community as a unit of identity , responsibilization and autonomization of people who reside in certain neighbourhoods , empowerment of individuals located in the margins , and expert management and outreach in risk communities. despite a seemingly emancipatory or rights - based agenda and the will to empower individuals who have been traditionally labelled as hard to reach ( patton 2011 ) , the deployment of these initiatives is indicative of the very form of government that miller and rose ( 2008 ) describe : the subject constituted through techniques of empowerment is being shaped into a better functioning and more responsible neoliberal actor empowered to make better decisions , able to fulfill moral obligations , and capable of committing to the health of his community . the experts are working directly in the community , relaying messages to individuals who have to be made aware of their personal allegiances to particular the community , as imagined through community viral load and the distribution of bodies in space , is now something to be programmed , researched and managed . the use of the term community is a powerful rhetorical tool and appeals to a common sense of belonging indeed , do we not all belong to some community ? yet , public health is not interested in all communities equally . as mentioned earlier , community viral load is part of a larger public health programme to promote high impact prevention using combinations of scientifically proven , cost - effective , and scalable interventions targeted to the right populations in the right geographic areas ( centers for disease control and prevention 2011 , p. 1 ) ( italics added for emphasis ) . within this new prevention framework , public health agencies are asked to identify communities where concentrations of the virus overlap with concentrations of substance users , gay and other men of have sex with men , transgender persons and racialized persons . community viral load provides a new tool for public health authorities to imagine particular communities ( legg 2005 ) in ways that are flexible , fluid , and can be adapted to different needs ( brown and knopp 2006 ) . this is inclusive of virtual communities of individuals who share particular risk factors but are geographically dispersed to communities of individuals who share geographical coordinates and whose virus is unsuppressed. what is important to understand here is not just that community viral load allows for these communities to be imagined , but that it is part of a large - scale operation to seek , test and treat more aggressively in target areas with the highest community viral load . this is part of the inner workings of community viral load ; it generates new knowledge , maps the presence of the virus collectively , makes room for a new form of surveillance , and in the end allows for the creation of specific targets , which are clustered around predominantly impoverished and marginalized neighbourhoods . seek , test and treat initiatives are arguably the best examples of high impact prevention efforts that specifically target such neighbourhoods and use on - site outreach to implement more aggressive forms of testing , surveillance and treatment -including mass testing fares with incentives meant to draw in the most marginalized . the rationale for these initiatives is largely based on the need to know community viral load and its geographic distribution . these initiatives appeal to the discourse of community ( lynn 2006 ) , with its shared culture , needs , and responsibilities , and relations of expertise ( miller and rose 2008 ) . we are particularly interested here in how community viral load creates or builds on existing personal allegiances and specific ties between individuals and communities to support the implementation of seek , test and treat initiatives ( miller and rose 2008 ) . not only is community viral load a potent tool to re - configure space ( as previously discussed ) and justify the need for public health to intervene more aggressively in certain neighbourhoods and locations , but it is also used as a device of identification . on the one hand , it concerns the identification of individuals who reside in areas where hiv is most heavily concentrated as members of a community. on the other hand , it reinforces the personal allegiances that these individuals may have to this community based on their serological status , their viral load , or the fact that they share a common fate as residents in neighbourhoods and locations where hiv is these individuals may , in turn , become actively involved in the deployment of community - level initiatives and engaged in outreach efforts to scale up hiv prevention . they may also become increasingly politicized . while this phenomenon may be beneficial for particular communities , it also tends to extend and reinforce the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 ) . seek , test and treat initiatives rely on the assumption that individuals who are made aware of their personal allegiances with a community will be more inclined to enrol , mobilize and act more responsibly ( miller and rose 2008 ) . one way to practice active personal responsibility is to take part in testing and link with prevention services . another way to practice active personal responsibility is to initiate antiretroviral treatment as soon as possible after diagnosis and demonstrate optimal treatment adherence . with this seek , test and treat initiatives make room for new relations of expertise and new opportunities for experts to intervene directly in the community. in fact , these initiatives rely on a re - configuration of space and re - location of interventions traditionally done in clinical settings ; testing is now conducted in vans , shelters , community centres , gay bathhouses and drop - in services , results are communicated right away , and linkage to care is automatically provided , surveillance data are broadened and include geospatial analysis , individual viral load , census information and indicators of socioeconomic status , and treatment is initiated rapidly after diagnosis followed by ongoing follow - up and adherence counselling in the community . they also rely on the ongoing presence and visibility of experts in the community , working in neighbourhoods and locations that are most affected by the hiv / aids epidemic . again , this may be beneficial for some communities but it is important to acknowledge that the expansion of testing and treatment coverage are not substitute to the removal of vulnerabilities that place people at risk of infection in the first place ( which incidentally , overlap with vulnerabilities preventing access to [ hiv care and ] treatment ( nguyen et al . 2011 , p. 292 ) . while our goal is not refute the scientific evidence in support of high impact prevention , which is the guiding framework for seek , test and treat initiatives , we consider that these programmes require more reflection . in this article , we have acknowledged that this new framework is closely tied to the introduction of community viral and the need for more effective ways to govern bodies that are seen as contributing to the spread of hiv . in light of our analysis , we consider that this new framework has very little to do with the care of individuals who live with hiv/ aids and much more to do with the production of a healthy population and the control of certain bodies . it is concerned with matters of life and death , with health and illness , with infectiousness and transmissibility , and with the collective processes that contribute to the spread of hiv . it acts upon the health of the population as a whole by targeting geographical areas where hiv is highly concentrated , seeking and testing populations conceived as inherently risk - prone , vulnerable or unstable , and treating individuals who are found to be hiv positive in order to achieve individual viral suppression . therefore , in keeping with foucault 's ( 2003 ) work , we consider that community viral load has both individualizing and massifying effects . it was introduced to the field of hiv as a way to act upon communities who are located in specific geographical areas rather than individuals themselves . however , in order for it to be effective , it has to create specific ties between individuals who reside in areas where hiv is most heavily concentration and their respective risk communities. this has important implications for people living with hiv / aids , and may have broader implications for everyone who is being forced into these particular geographical areas these new ghettos that are marked by hiv and are under the radar of public health authorities . as such , we are concerned that community viral load has become a proxy for naming risk communities , but with new levels of sophistication . in particular , we are concerned with the potential to increase stigma directed at populations who occupy areas that are identified as highly virulent. this phenomenon has yet to be addressed in the literature and in research conducted on community viral load . the objective of this article has been to formulate a critique and propose a novel way of theorizing community viral load through a foucauldian analytics of governmentality with an attention to the role of space and community . we have shown that particular kinds of spaces are being reconfigured through a combination of clinical measures and epidemiological techniques to map the distribution of infected and risky bodies . these spaces then become key targets of public health intervention in the form of high impact treatment and prevention technologies that necessitate an unprecedented investment in identifying individuals in need of treatment ( nguyen et al . once on treatment , individuals become part of the community viral load loop their individual viral load becomes used to assess their we have also shown that the term community and its uses represents a discursive terrain imbued with power and ideology and requires unpacking to surface how it is being used and to what ends ( lynn 2006 ) . in light of our analysis , we are struck by what appears to be a shift back to the beginnings of the aids epidemic when various groups were singled out as infectious ( novitsky and essex 2012 ) and characterized as dangerous [ for historical overview and critique of this , see epstein 1996 , patton 1996 ] . the concern became that identifying people in this way perpetuated stigma and could result in the potential for an even greater number of infections ( parker and aggleton 2003 , peretti - watel et al . , epidemiology moved to monitoring risk behaviours as a way of de - stigmatizing these groups . with the invention and deployment of community viral load , we see a return to this previous logic , but resting on the seeming neutrality of space as if all people freely inhabit spaces of their own choosing . the implications of this , some of which we have explored , are only speculative at this time as calculating community viral load in all jurisdictions has various logistical and policy challenges . there are however concerted efforts underway to reduce these remaining barriers as suggested by the most recent national hiv / aids strategy in the united states ( office of national aids policy 2010 ) and guidelines published by the centers for disease control and prevention ( 2012 ) on community viral load measures , definitions and methods for calculation . this return to previous ways of thinking about , and managing hiv , albeit through more sophisticated techniques than were available at the beginnings of the epidemic , raises important questions about how hiv is being seen by governments , policy makers and public health agencies . require testing and mandatory treatment on a scale seen only in dictatorships ( p. 263 ) . however , this bold statement fits within larger debates about hiv as a threat to inter / intra state security ( elbe 2005 , 2009 ) . how do the changing politics of hiv and the growing interest in the securitization of health and illness help inform our thinking about mapping community viral load what are the implications of these clinical advancements for other health issues , and the potential outcomes to those who reside in spaces characterized by high rates of hiv and other forms of chronic illness . we hope that by closing on such a note that others will be encouraged to revisit hiv , and these new treatment and new prevention technologies , as a site for critical debate .

[ purpose ] the purpose of this study is to investigate the effects of virtual reality training using nintendo wii on balance and walking for stroke patients . [ subjects and methods ] forty stroke patients with stroke were randomly divided into two exercise program groups : virtual reality training ( n=20 ) and treadmill ( n=20 ) . the subjects underwent their 40-minute exercise program three times a week for eight weeks . their balance and walking were measured before and after the complete program . we measured the left / right weight - bearing and the anterior / posterior weight - bearing for balance , as well as stance phase , swing phase , and cadence for walking . [ results ] for balance , both groups showed significant differences in the left / right and anterior / posterior weight - bearing , with significant post - program differences between the groups . for walking , there were significant differences in the stance phase , swing phase , and cadence of the virtual reality training group . [ conclusion ] the results of this study suggest that virtual reality training providing visual feedback may enable stroke patients to directly adjust their incorrect weight center and shift visually . virtual reality training may be appropriate for patients who need improved balance and walking ability by inducing their interest for them to perform planned exercises on a consistent basis . hiv plasma viral load testing has become more than just a clinical tool to monitor treatment response at the individual level . increasingly , individual hiv plasma viral load testing is being reported to public health agencies and is used to inform epidemiological surveillance and monitor the presence of the virus collectively using techniques to measure community viral load. this article seeks to formulate a critique and propose a novel way of theorizing community viral load . based on the salient work of michel foucault , especially the governmentality literature , this article critically examines the use of community viral load as a new strategy of government . drawing also on the work of miller and rose , this article explores the deployment of community through the re - configuration of space , the problematization of viral concentrations in specific microlocales , and the government ( in the foucauldian sense ) of specific bodies which are seen as risky , dangerous and therefore , in need of attention . it also examines community viral load as a necessary precondition forming the conditions of possibility for the recent shift to high impact prevention tactics that are being scaled up across north america .

endovascular aortic aneurysm repair ( evar ) has developed into a feasible and successful alternative to open surgery for the treatment of abdominal aortic aneurysms . evar can be offered to many patients with a suitable anatomy of the aorta and iliac arteries , regardless of comorbid conditions [ 13 ] . despite the known excellent early results of evar in terms of the reduction in perioperative mortality , rate of complications and length of hospitalization , many patients require re - intervention during the middle and long - term follow - up because of procedure - related complications . for this reason , surveillance of these patients is crucial to determine the long - term performance of these devices [ 4 , 5 ] . because of the rapid diffusion of evar and the increased number of patients who undergo multidetector ct ( mdct ) follow - up , the radiologist should be familiar with the full spectrum of possible procedure - related complications in order to allow their early diagnosis and treatment . the purpose of this article is to present a spectrum of post - evar complications as seen with mdct . regardless of the scanner available ( 4 , 16 or 64 row ) , a thick - slice unenhanced acquisition can be performed to visualize the position of the stent - graft to reveal calcifications and to plan the following contrast - enhanced examination by selecting the acquisition volume and placing a region of interest in the aorta at the level of the celiac trunk ( if bolus - tracking software is used ) . contrast - enhanced images in the arterial phase are obtained during bolus intravenous injection of 90130 ml of iodinated high - concentration non - ionic contrast medium , administrated with an automated injector at a flow rate of 34 ml / s through an antecubital vein . ct acquisition protocol parameters and scanning coverage ranges are illustrated table 1 and fig . 1 . scanning should start when the examined structures have reached an ideal level of opacification ; therefore , the scan delay must be individualized to the patient by using bolus - tracking software to capture 100 hu on the abdominal aorta . at the end of the arterial acquisition , delayed images focused on the graft must be acquired , performed at least 60 s and up to 120 s after contrast material injection . the cta examination can be complemented by postprocessing reconstructions , including maximum- intensity projection ( mip ) , curvilinear reformation ( cvr ) and volume rendering ( vr ) .table 1ct acquisition protocol parametersscannerrotation time ( s)collimationtable feed ( mm / s)slice thickness ( mm)slice interval ( mm)duration ( s)4 slice0.54 1 mm251.25125 - 3016 slice0.516 0.625 mm27.50.6250.62525 - 3064 slice0.564 0.625 mm800.6250.625<15fig . 1scanning coverage of unenhanced and enhanced ct - acquisition ct acquisition protocol parameters scanning coverage of unenhanced and enhanced ct - acquisition an aortic stent - graft is a device composed of a metallic portion ( nitinol , elgiloy , and stainless steel ) and graft material ( polyester , ptfe ) . on ct images only the metallic portion is visualized . on the basis of the general morphology , there are three types of stent - grafts available for treating abdominal aortic aneurysms : straight , aorto - uni - iliac grafts and bifurcated . straight aortic tube grafts have the proximal and distal attachment sites in the aorta , above the aortic bifurcation ( aorto - aortic ) . the aorto - uni - iliac device is a stent - graft that is deployed from the supra - aneurysmal aorta to one iliac artery only ; the opposite iliac artery is then occluded with an endovascular occlusion device in order to prevent retrograde blood flow into the aneurysm sac , and a femoro - femoral crossover graft maintains blood flow into the opposite limb ( fig . 2 ) . bifurcated stent - grafts are extended to the iliac arteries . on the basis of proximal fixation , the suprarenal fixation device has an uncovered metallic portion placed above the ostia of the renal arteries and a covered portion placed below the renal arteries : the radiological markers placed between the uncovered and covered portion are represented by two metallic points located on the stent - graft metallic structure at the opposite position [ fig . the fenestrated device has a covered metallic portion of the graft that incorporates any visceral vessels such as the renal arteries , superior mesenteric artery or celiac trunk ; the patency of the incorporated vessels is maintained by the associated placement of covered stents ( fig . 2vrt ( a ) and mip ( b ) images : aorto - uni - iliac device associated with a surgical femoro - femoral cross - over graft . 3scout view and axial images showing the two metallic radiological markers placed in the opposite site between the uncovered and covered portion in a suprarenal fixation devicefig . 4mip ( a ) and vrt ( b ) images show a fenestrated stent - graft with a covered metallic portion that incorporates the renal arteries , which are made patent by the associated placement of covered stents ( c ) vrt ( a ) and mip ( b ) images : aorto - uni - iliac device associated with a surgical femoro - femoral cross - over graft . the left iliac limb is occluded by an occluder ( arrows ) scout view and axial images showing the two metallic radiological markers placed in the opposite site between the uncovered and covered portion in a suprarenal fixation device mip ( a ) and vrt ( b ) images show a fenestrated stent - graft with a covered metallic portion that incorporates the renal arteries , which are made patent by the associated placement of covered stents ( c ) stent - graft and distal branches are considered patent if a uniform and homogeneous contrast enhancement is detected within them . on the other hand , graft thrombosis is recognized as an intraluminal , concentric or eccentric hypodense area within the stent - graft on ct images obtained after contrast - media injection . angulation of the prosthesis could lead to a stenosis or thrombosis with consequent reduction of either the inflow or outflow , with increased possibility of in - stent thrombosis and stenosis , which can be manifest in about 319 % of cases [ 8 , 9 ] . graft occlusion appears as a complete non - enhancing intraluminal area within the stent - graft . graft limb occlusion may be directly due to compression of the limb in the proximal neck or in a narrow distal aneurysm neck , or to kinking or compression of the limb in a narrow or tortuous iliac artery . in case of iliac limb thrombosis , a femoro - femoral cross - over graft can be made , whereas a main body occlusion needs a complete surgical conversion . 5axial ( a ) and cpr ( b c ) images show a stent graft thrombosis causing significant intraluminal stenosis axial ( a ) and cpr ( b c ) images show a stent graft thrombosis causing significant intraluminal stenosis fractures or distortions of the metallic stent - graft structure are rare but important complications : these complications are better visualized on 3d rather than axial images ( fig . another complication that must be promptly recognized is stent - graft migration that occurs because of poor attachment of the stent to the aortic wall , which can cause sac reperfusion and subsequent aneurysm rupture.fig . 6mpr ( a ) and mip ( b ) images show a distortion of the metallic stent - graft structure . the integrity of the graft material as demonstrated by the linear and defined margin of the contrast - medium inside the stent - graft needs to be observed ( arrows ) . a type - ii endoleak is also detected ( * in b ) mpr ( a ) and mip ( b ) images show a distortion of the metallic stent - graft structure . the integrity of the graft material as demonstrated by the linear and defined margin of the contrast - medium inside the stent - graft needs to be observed ( arrows ) . unless the graft can be extended proximally , treatment is usually carried out by conversion to open repair . only migration of more than 4 mm this is related to a number of factors , including inadequate proximal fixation ( incorrect sizing , conical - shaped neck , short neck and angulation of the neck ) , progressive dilatation of the proximal neck and aneurysm size , as well as iliacal fixation [ 1012 ] . mdct angiography clearly detects minimal stent - graft migration : the diagnosis is based on the comparison of 1-month follow - up ct images evaluating the relationship between the proximal end of the stent - graft and lumbar vertebral body or renal artery origin on 3d and axial images , respectively ( fig . 7).fig . 7one month ( a ) and 1 year ( b ) post - evar follow - up sagittal mip images . at 1 year follow - up the stent graft appeared to have migrated distally with a significant angulation of the main body one month ( a ) and 1 year ( b ) post - evar follow - up sagittal mip images . at 1 year follow - up the stent graft appeared to have migrated distally with a significant angulation of the main body since aneurysm exclusion is expected to be correlated with shrinkage , surveillance of aneurysm dimensions is mandatory for asserting the adequacy of aneurysm exclusion from the blood flow and for guiding the need for reintervention in selected cases . the aneurysm volume may increase slightly immediately after evar ( 34 mm ) , given the additional volume and the external force exerted by the stent - graft or , alternatively , the swelling of the aneurysm sac induced by perigraft thrombosis . on the other hand , the volume tends to increase if sac perfusion is present or decreases during the follow - up in the absence of endoleaks . diameter measurements are most accurate if performed on axial images perpendicular to the aortic long axis , but volume assessment has been proven to be more accurate than diameter in the early detection of aneurysm growth ; however , volume assessment is time - consuming and requires advanced processing , dedicated equipment and skilled operators . endoleak is defined as a persistent blood flow within the sac excluded from the stent - graft , and it occurs in 2.445.5 % of patients after evar . on cta images , the endoleak appears as a high attenuation area outside the graft but within the aneurysm sac , detected on arterial and/or delayed phase images , but generally absent on unenhanced images . unenhanced images can be helpful to avoid false - positive diagnoses , allowing the differentiation of calcifications in the aneurysm sac from an endoleak ( fig . because endoleaks have variable flow rates , they can be detected at variable times after contrast material injection . for this reason , a delayed phase has been recommended : in detail , this phase could detect endoleaks not visualized during the arterial phase , the so - called low - flow endoleaks . early endoleaks occur in the first 30 days following evar , while endoleaks that fail to seal within 30 days are called persistent endoleaks . proper classification of an endoleak is important for its subsequent management [ 16 , 17].type i is caused by separation of the device from the arterial wall , resulting in leaks originating at the proximal and/or distal attachment sites of the graft because of a technical ( e.g. , suboptimal stent - graft diameter ) or anatomical ( e.g. , a short , irregular , ulcerated or angulated landing zone without an optimal conformation of the stent - graft to the curved aortic contour ) problems , or to its caudal migration . on cta images , it often appears as a huge and circumferential leak , adjacent to the proximal or distal end of the prosthesis ( figs . 9 and 10).type ii endoleaks are caused by back - filling of the aneurysm sac via branch vessels , such as the lumbar arteries and inferior mesenteric artery excluded by the stent - graft . on cta images , the type ii endoleak is most pronounced at the periphery of the aneurysmal sac , with little or no contact with the prosthesis , is commonly located in a posterior or lateral position , and is associated with opacification of the lumbar arteries . if an endoleak is located in the anterior position , a retrograde flow into the sac by the inferior mesenteric artery must be suspected ( fig . 11).type iii endoleaks arise from a fabric tear , modular or graft disconnection , and are more likely when multiple prostheses with short overlapping areas are used . on cta images , the leak is strictly adjacent to the prosthesis , with little or no contact with margins of the aneurysmal sac , without opacification of the lumbar arteries or inferior mesenteric artery ( fig . 12).type iv involves vascular flow caused by the high porosity of the graft , most likely created by the numerous suture holes holding the graft material to the stent . they are usually only detected on conventional angiograms performed at the end of the procedure.type v or endotension refers to a growth of the aneurysm sac but without demonstrable reperfusion defects ( fig . the combined analysis of unenhanced ( a ) , arterial ( b ) and late - phase enhanced images ( c ) allows the correct characterization of the thin hyperdensity within the sac ( arrows ) as a linear calcificationfig . 9axial ( a ) and mip- ( b ) images show the stent - graft caudal migration with subsequent proximal type - i leak ( * ) fig . 10axial ( a ) and cpr ( b ) images show an incomplete distal attachment of the right iliac limb causing a type i leak , as confirmed by dsa ( c)fig . axial ( a ) and thin mip ( b , c ) images show back - filling of the aneurysm sac through the inferior mesenteric arteryfig . 12vrt ( a , b ) , mip ( c ) and cpr ( d ) images show a type iii endoleak due to a right iliac limb disconnection and distal migrationfig . follow - up mdct examinations performed at 6 months ( a ) and 12 months ( b ) show an increase in sac diameter without evidence of endoleaks type i is caused by separation of the device from the arterial wall , resulting in leaks originating at the proximal and/or distal attachment sites of the graft because of a technical ( e.g. , suboptimal stent - graft diameter ) or anatomical ( e.g. , a short , irregular , ulcerated or angulated landing zone without an optimal conformation of the stent - graft to the curved aortic contour ) problems , or to its caudal migration . on cta images , it often appears as a huge and circumferential leak , adjacent to the proximal or distal end of the prosthesis ( figs . 9 and 10 ) . type ii endoleaks are caused by back - filling of the aneurysm sac via branch vessels , such as the lumbar arteries and inferior mesenteric artery excluded by the stent - graft . on cta images , the type ii endoleak is most pronounced at the periphery of the aneurysmal sac , with little or no contact with the prosthesis , is commonly located in a posterior or lateral position , and is associated with opacification of the lumbar arteries . if an endoleak is located in the anterior position , a retrograde flow into the sac by the inferior mesenteric artery must be suspected ( fig . type iii endoleaks arise from a fabric tear , modular or graft disconnection , and are more likely when multiple prostheses with short overlapping areas are used . on cta images , the leak is strictly adjacent to the prosthesis , with little or no contact with margins of the aneurysmal sac , without opacification of the lumbar arteries or inferior mesenteric artery ( fig . type iv involves vascular flow caused by the high porosity of the graft , most likely created by the numerous suture holes holding the graft material to the stent . they are usually only detected on conventional angiograms performed at the end of the procedure . type v or endotension refers to a growth of the aneurysm sac but without demonstrable reperfusion defects ( fig . the combined analysis of unenhanced ( a ) , arterial ( b ) and late - phase enhanced images ( c ) allows the correct characterization of the thin hyperdensity within the sac ( arrows ) as a linear calcification axial ( a ) and mip- ( b ) images show the stent - graft caudal migration with subsequent proximal type - i leak ( * ) axial ( a ) and cpr ( b ) images show an incomplete distal attachment of the right iliac limb causing a type i leak , as confirmed by dsa ( c ) type ii endoleak . axial ( a ) and thin mip ( b , c ) images show back - filling of the aneurysm sac through the inferior mesenteric artery vrt ( a , b ) , mip ( c ) and cpr ( d ) images show a type iii endoleak due to a right iliac limb disconnection and distal migration type v endoleak ; axial images . follow - up mdct examinations performed at 6 months ( a ) and 12 months ( b ) show an increase in sac diameter without evidence of endoleaks type i and type iii endoleaks require prompt treatment as they are associated with a high risk of sac rupture . however , the most common endoleak found in endovascular stent - grafting is the type ii endoleak . renal artery stenosis , occlusion and dysfunction represent the main concern with evar , especially in patients with inadequate anatomy of the proximal aneurysm neck , pre - existing renal disease or in whom suprarenal fixation was used . axial ct images are commonly used to monitor the patency of these arteries following stent - graft placement and to detect renal infarction following suprarenal stent grafting . however , both mip and cpr are useful and complementary to axial images for the detection of stenosis and occlusions because of the tortuous course of the renal arteries . the additional views provided by ct angiography allow displaying the renal arteries in multiple planes and projections , which is often necessary for depiction of stenosis . in renal infarction , contrast - enhanced ct scans demonstrate a sharply demarcated , wedge - shaped area of decreased attenuation in the kidney ( fig . however , to date no significant correlation has been found between evar and renal impairment [ 1920].fig . the right kidney appeared decreased in size and showed an area of parenchymal hypoperfusion due to infarction mip images obtained before ( a ) and after ( b ) evar . the right kidney appeared decreased in size and showed an area of parenchymal hypoperfusion due to infarction usually a transfemoral approach is used to perform evar , exposing the femoral artery by a vertical groin incision . the large - caliber delivery sheaths used for evar , up to 26 fr , increase the morbidity rate compared to the usual catheter size used for routine diagnostic angiography . the minimum vessel diameter required to allow passage of the evar device is 78 mm . access site complications include dissection , demonstrated by a linear endoluminal hypodense area ( intimal flap ) ( fig . 15a b ) or arterial rupture , with contrast material extravasation outside the artery . other complications are pseudoaneurysms , especially close to the entrance site , hematomas near the femoral incision , demonstrated by a high - attenuating area ( fig . 15c ) , infections and lymphoceles .fig . 15axial ( a ) and mip ( b ) images show an endoluminal hypodense line within the right external iliac artery representing a dissection ; c axial image shows bilateral small hematomas at the femoral incision site axial ( a ) and mip ( b ) images show an endoluminal hypodense line within the right external iliac artery representing a dissection ; c axial image shows bilateral small hematomas at the femoral incision site mdct is the method of choice both in the pre- and postoperative setting of evar procedures . a better understanding of the procedure followed , the devices used , the normal postoperative imaging features and the possible procedure - related complications ensures an optimal planning and follow - up of patients who undergo an evar procedure . finally , in order to better determine the long - term performance of these devices , strict surveillance of all patients is mandatory this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited . multiple sclerosis ( ms ) is considered a chronic inflammatory disease of the central nervous system of autoimmune origin . onset of disease is most frequently in young adulthood , between 20 to 40 years of age.1 although many aspects of ms pathogenesis have been elucidated , the exact causal mechanisms are still not fully understood . an interplay between environmental factors in genetically susceptible individuals is assumed.2 this concept is supported by a wealth of research findings.35 unambiguously , once the disease has developed , it continues lifelong , and there is still no cure . the course of the disease can be relapsing remitting , which means that episodes with exacerbation of neurological symptoms alternate with periods of remission.6 over time , these relapses often do not fully resolve , leading to a stepwise accumulation of disability . moreover , after 1015 years , one - half of patients with initial relapsing remitting disease course will develop a secondary progressive disease7 that is characterized by a progressive increase of disability , independent of relapses and predominantly caused by deterioration of walking ability . only about 10%15% of patients exhibit a primary progressive disease course , which is defined by at least one year disease progression from onset on.8 a clinically isolated syndrome ( cis ) is diagnosed in patients with a first clinical event suggestive of ms and evidence of disease dissemination in space , but not yet evidence of dissemination in time , thus chronicity of disease . one of the hallmarks of ms is a high variability of the natural disease course . while some patients , apart from relapses , never experience any handicap , the majority of patients will ultimately develop disability to a variable degree and after a variable time interval after onset of disease . as first shown by confavreux et al9 and based on a concept of a two - stage disease with focal inflammation in the early stage and diffuse inflammation and neurodegeneration in a second - disease stage , leray et al recently confirmed highly interesting data on time to disability accumulation in ms:10 while duration of the first stage from onset of disease until reaching a milestone of kurtzke disability status scale ( dss ) 311 ( corresponding to moderate disability ) ranged from less than 3 years to more than 15 years , duration of phase ii ( kurtzke dss 3 to dss 6 , the latter is defined by requiring unilateral assistance to walk 100 meters ) remained nearly identical in all patients with 69 years , irrespective of the duration of phase i. this observation suggests that , once a clinical threshold of irreversible disability is reached , further progression of disability becomes inevitable . support of this hypothesis can be derived from two typical findings of the chronic progressive phase of ms . even in the absence of new or contrast - enhancing magnetic resonance imaging ( mri ) lesions , disability usually increases in the chronic progressive phase of the disease , and once the chronic progressive phase is reached , efficacy of disease - modifying therapies often declines.12,13 although relapse rate and progression of the expanded disability status score ( edss)11 are widespread parameters of disease activity , many patients with ms do not only suffer from physical disability ; they also suffer from neurocognitive decline , fatigue , or depression.14,15 hence , the diagnosis of ms is a life - changing event with a significant impact on family , society , and the social welfare system . fortunately , during the last decades , several disease - modifying therapies ( dmt ) have been licensed that ameliorate the course of the disease.16 the aim of these dmt is to reduce inflammation , disease activity as measured by mri , and relapse rate . thereby , a delay of transition into the secondary progressive phase and prevention of permanent disability ought to be achieved . due to the chronic nature of ms , dmts are usually applied continuously over many years , or even decades . to keep in mind , dmts are applied in a preventive manner , since neuronal damage can not be reversed . ms is the most frequent nontraumatic cause for disability among young adults in north america and europe.17 worldwide , about 2.5 million people are affected by ms ; although traditionally , the incidence is supposed to vary by geographical latitude.18 the highest prevalence with more than 30 ms patients per 100,000 inhabitants can be found in northern europe and north america . a medium prevalence of 530 per 100,000 is observed in southern europe and the southern united states ; whereas , a low prevalence of fewer than 5 per 100,000 has been reported for asia and south america.19 population genetics , the geographically determined physical environment , and socioeconomic structures have been proposed as possible reasons for continental differences in ms prevalence.20 in the united states alone , about 400,000 individuals suffer from ms ; each week , more than 200 persons are newly diagnosed with the disease.18 according to estimates for 2010 , ms has been diagnosed in 540,000 subjects of the european population.21 however , recent epidemiological studies found an increasing incidence and prevalence of ms , especially in women.20 this seems to be the case also in areas of the world that were formerly classified as low - prevalence regions . for example , ms prevalence was recently reported as 3155 per 100,000 inhabitants in the arabian gulf region and 0.8321.5 per 100,000 individuals in latin america and the caribbean.22,23 factors contributing to an increasing incidence of ms have not been fully elucidated . earlier ms diagnosis , due to revised diagnostic criteria ; better ascertainment of diagnosis ; less sunlight exposure ; higher standards in hygiene ; and cigarette smoking , have all been proposed as likely candidates.20,24 the economic impact of ms can be divided into direct costs and indirect costs . direct costs account for health care costs , such as pharmaceuticals , inpatient care costs , outpatient care costs , additional treatments like physiotherapy , and medical aids . in contrast , indirect costs are caused by productivity loss of patients and caregivers.25 this might be due to sick leave , reduced daily working time , unemployment , or premature retirement . in europe , mean annual cost per person with ms is estimated to be around 27,000 , ranging from 7,227 in bulgaria to 44,565 in luxembourg . on average , about two - thirds of the totals are caused by direct costs , while approximately one - third are derived from indirect costs.21 in 1998 , all - over costs of ms in the united states were us$6.8$11.9 billion annually , which averages about us$34,000 per patient , per year.26 in a survey among patients receiving dmt in the united states in 2004 , total average costs per patient and year were estimated as high as us$47,000.27 of these , 63% accounted for direct costs , including 34% of total costs ( us$16,000 ) for dmt . of note , patients having suffered a relapse were found to have higher costs than patients without relapse.27,28 furthermore , several studies have shown that total costs rise with increasing disability.2832 in line with this result , a secondary and primary progressive disease course was found to be more costly than a relapsing remitting one , due to a rise of indirect costs . besides degree of physical disability , age , depression , and fatigue have been identified as independent factors of an increase of costs.32 quality of life remarkably and constantly declines with advancing stages of disability , and the occurrence of relapses negatively affects quality of life measures.28,31 it was in 1993 that interferon beta-1b was introduced as the first dmt of ms . still , it represents a very common first - line treatment that is widely used . remitting ms , and secondary progressive ms with superimposed relapses.33 there are three different recombinant interferon beta preparations on the market . interferon beta-1b ( betaferon , bayer healthcare pharmaceuticals , berlin , germany ; betaseron , bayer healthcare pharmaceuticals , berlin , germany ; extavia , novartis , basel , switzerland ) is recombinantly expressed in escherichia coli and is subcutaneously injected every other day . interferon beta-1a is recombinantly expressed in chinese hamster ovary cells and is identical to the human interferon beta.34 there are two preparations of interferon beta-1a . rebif ( merck serono , geneva , switzerland ) is subcutaneously administered three times a week . avonex ( biogen idec , cambridge , ma , usa ) is applied once weekly as an intramuscular injection . placebo - controlled trials have demonstrated a significant reduction of disease activity for all three interferon beta preparations.3537 the pivotal trial on efficacy of interferon beta-1b in ms , which led to the initial approval of the drug for treatment of ms , demonstrated an annual relapse rate of 1.27 for placebo and 0.84 for 250 g interferon beta-1b.35 similar results were observed for interferon beta-1a . in the prevention of relapses and disability by interferon beta-1a subcutaneously in multiple sclerosis ( prisms ) trial on subcutaneous interferon beta-1a , the annual relapse rate was 1.28 for placebo , 0.91 for 22 g , and 0.87 for 44 g.37 intramuscular interferon beta-1a reduced the annual relapse rate to 0.61 , compared with 0.90 in the placebo group.36 these findings are supported by significant reduction of mri disease activity in all three trials , while only in the latter two trials , a beneficial effect on disability progression was recorded during this 2-year study period . while these therapies have been introduced , a better understanding of ms pathophysiology especially the knowledge of unrecoverable axonal loss already in early stages of the disease has prompted a fundamental shift in the ms treatment approach and provided the rational for early ms treatment.33,34 based on this consideration , dmts are initiated early in the course of the disease and before irreversible neuronal damage and disability have occurred . following the concept of early ms treatment , interferon beta treatment has been investigated in patients with cis and has shown a beneficial effect in terms of conversion to definite ms , edss progression , and mri disease activity.3841 in the betaferon/betaseron in newly emerging multiple sclerosis for initial treatment ( benefit ) trial,40 patients with cis were randomized to receive interferon beta1-b or placebo for 2 years , or until clinically definite multiple sclerosis ( cdms ) was diagnosed , according to the poser et al criteria.42 within the 2-year study period , 45% of patients in the placebo group versus 28% of the interferon beta group progressed to cdms , according to poser et al ; and 85% of the placebo versus 69% of the interferon beta group were diagnosed with definite ms , according to the mcdonald criteria of 2001.43 both results reached statistically significant difference . these data revealed a significant lower number of newly active lesions , cumulative number of new t2-weighted mri lesions and gadolinium - enhancing lesions , as well as cumulative volume of gadolinium - enhancing lesions in the interferon beta - treated patients group . of note , there was no change of health - related quality of life observed during the 2 years . after having finished the placebo - controlled core study , patients were offered to enroll in an open - label interferon beta-1b 250 g follow - up trial to investigate effects of early treatment in cis , versus delayed treatment after diagnosis of cdms or after 2 years on study . three years after initial randomization , 37% of the early treatment group versus 51% of the delayed - treatment group have developed cdms ( risk reduction of 41% ) ; 16% of the early treatment group versus 24% of the delayed - treatment group experienced confirmed edss progression ( risk reduction 40%).44 a subgroup analysis revealed lower treatment effects in patients with less clinical or mri activity at the time of the first event ; patients with initial multifocal presentation and high lesion load would benefit more from early treatment . in the 5-year active treatment extension follow - up study , risk reduction of cdms remained 37% in the early treatment group ; whereas , no statistically significant difference in confirmed disability progression was observed any more between early and delayed interferon beta treatment.45 in both treatment groups , median edss remained 1.5 during the 5-year study period . finally , in the benefit extension trial , a long - term international observational study of patients having completed the initial placebo - controlled core study , data on follow - up for more than 8 years are available.46 at the discretion of the physician and according to local standards , patients were treated with any of the disease - modifying drugs or received no treatment . during the study periods , 5.1% of patients received no treatment ; 77.6% received no other drug than interferon beta-1b ; 6% , interferon beta-1a ; 5.8% , glatiramer acetate ; and 6.6% of patients received any escalation therapy . in the overall study population , disability as quantified by the edss changed only slightly over 8 years , with the median edss remaining 1.5 at year 8 . however , 55.5% of patients , who had received early interferon beta treatment in the core study , developed clinically definite ms , while clinically definite ms was diagnosed in 65.8% of patients with delayed interferon beta treatment . moreover , a difference of the overall annualized relapse rate was still observed over the 8-year period 0.20 early treatment versus 0.26 delayed treatment.47 placebo - controlled clinical trials on interferon beta-1a in cis have yielded very similar results,38,41 suggesting a class effect of interferon beta treatment in early ms and cis . by numerous investigational trials and the clinical experience in interferon beta for two decades , safety and tolerability profile of this drug is well - known and has proven favorable , also , in long - term application . side effects mainly include injection site reactions , flu - like symptoms , leukopenia , and liver - enzyme elevation.48 moreover , up to 40% of patients develop antibodies against interferon beta , which antagonize bioactivity in a significant proportion of patients.4951 if neutralizing antibodies against interferon beta persistently occur , therapeutic efficacy of the drug is reduced or abolished.5256 besides interferon beta glatiramer acetate ( [ ga ] , copaxone , teva , petah tikva , israel ) represents a common first - line therapy with daily subcutaneous injections , which is approved for treatment of patients with relapsing remitting ms , as well as for patients with cis and typical mri features of ms . the precise ( early glatiramer acetate treatment in delaying conversion to clinically definite multiple sclerosis in subjects presenting with a clinically isolated syndrome ) study demonstrated that ga reduced the risk of developing clinically definite multiple sclerosis by 45% , compared with placebo during a 3-year study period.57 interferon beta and ga are the only agents that are licensed for treatment of cis . however , for the sake of completeness , it needs to be mentioned that there are several other therapeutics licensed for baseline and escalation therapy in definite ms , and more agents are likely to enter the market.16 approval of all these agents is usually based on efficacy and safety data of a 2-year clinical trial . in ms phase ii and phase iii interventional clinical trials , a duration of about 6 months to a few years has become common , since demonstration of efficacy usually needs several months of treatment . on the other hand , duration of clinical trials longer than 2 years will make a well - controlled trial less feasible to accomplish and might hold back an effective treatment from ms patient care . given an increasing number of available dmt in ms , trials investigating the long - term efficacy are needed . this is to briefly summarize the terms and types of pharmacoeconomic evaluations used in the studies reviewed here ( adapted from clifford goodman unless cited otherwise).25 cost - effectiveness analyses ( cea ) measure costs in monetary units and consequences in natural units , such as life - years gained , or relapses avoided.58 in cost - utility analyses ( cua ) , a variant of cea , consequences are measured in terms of preference - based measures of health,58 most commonly based on quality - adjusted life - years ( qaly).59 a qaly is a unit that adjusts gained or lost life - years subsequent to an intervention by the quality of life during those years . gained or lost life - years are multiplied by a weighting factor ( utilities ) ranging from 0.00 ( representing death ) to 1.00 ( representing a perfect health state ) . cea and cua always involve comparison of alternative interventions , expressed as incremental cost - effectiveness ratio ( icer ) and incremental cost - utility ratio ( icur ) , respectively . therefore , incremental costs are divided by the change of effectiveness or utility ( eg , qualys gained ) . depending on the perspective of pharmacoeconomic evaluations ( eg , society overall , third - party payers , patients ) , different types of costs are of interest . costs need to be corrected for effects of inflation ; both costs and outcomes should be discounted . discounting reflects the fact that costs and benefits tend to have less value in the future than in present . the former approach involves collection of original data ( eg , randomized controlled studies ) . quantitative modeling - based pharmacoeconomic analyses simulate costs and consequences of pharmacological interventions , based on existing data and estimates of key variables where there are no data available . in this type of analysis it is based on predefined sets of health states ( eg , edss levels ) . its time horizon is divided into equal increments of time , referred to as markov cycles . for each cycle , costs , outcomes , and transition probabilities of moving from one state to another or of remaining in the same state are estimated.60 finally , sensitivity analyses determine robustness of results of model - based analyses by varying the estimates of key variables within plausible ranges ( eg , by applying a multivariate approach ) . we conducted a systematic literature search in medline ( pubmed ) on december 14 , 2012 , applying the medical subject headings ( mesh ) terms multiple sclerosis and costs and cost analysis . we deliberately chose the comprehensive mesh term costs and cost analysis to cover all potentially relevant studies . four hundred fourteen publications met our search criteria , including 40 non - english publications , which were excluded ; titles and abstracts of the resulting 374 publications were reviewed by fc and db . inclusion criteria were as follows : ( 1 ) original research on the pharmacoeconomics of dmt in ms ; ( 2 ) comparative study ; and ( 3 ) comparators ( initiation of dmt at the time of the first demyelinating event versus treatment after cdms ) . because our search strategy resulted in no more than four relevant studies,6164 we did not exclude the study by curkendall et al,62 although the authors only report on costs and not on health - outcome measures of dmt in cis . the remaining three studies were rated for quality of health - economic analyses , based on the quantitative scoring system that has been proposed by chiou et al ( range 0100 ; 0 representing the lowest ; 100 representing the highest quality).65 the 16-item weighted scoring system was independently applied by fc and db . our scoring results were in accordance with the results reported by yamamoto and campbell , who chose the same approach ( table 1).66 as outlined before , early initiation of dmt with interferon beta or ga has been proven to reduce time to cdms , relapse rate , and disease progression . this observation led to the approval of interferon beta and ga for treatment of relapsing remitting ms , as well as cis . therefore , initiation of dmt may be started even before definite diagnosis of ms is established . to date , four studies have been published that address the economic impact of starting dmt at the time of the incident cis versus after conversion to cdms ( table 1).6164 the swedish study by caloyeras et al estimates cost - effectiveness of interferon beta-1b if initiated in cis compared with delayed treatment after the diagnosis of cdms has been made.61 the authors applied a markov model analysis , based on twelve health states defined by edss scores , the assigned diagnosis ( ie , cis versus cdms ) , and the course of disease ( ie , relapsing or nonrelapsing forms of ms , and death ) . transition probabilities after 6-month cycles were estimated for a lifetime time horizon of 50 years . the authors assumed that treatment discontinuation rates would not differ between both treatment arms ; all hypothetical patients would discontinue dmt after roughly 25 years . switching to another dmt and the core input data for the statistical model were derived from the benefit trial.40,44,45 moreover , extrapolations of benefit data and best available data from the published literature were used as model parameters . results of the cua demonstrate that , during a 50-year time horizon , patients assigned to the early treatment group gained 12.9 qalys , as compared with 12.4 qualys in the delayed - treatment group . on average , patients in the delayed - treatment group progressed 2 years earlier to cdms ( estimated conversion rate 99.54% ) , compared with patients in the early treatment group ( estimated conversion rate 99.31% ) . direct medical costs ( ie , interferon beta-1b drug costs , costs for inpatient and outpatient care , and costs related to testing , other drugs , and relapse events ) were higher in the early treatment arm , whereas indirect costs ( ie , costs related to early retirement and short - term absence ) and direct nonmedical costs ( ie , informal care , services , investments ) in the delayed - treatment group exceeded those in the early treatment group . when all costs were summed up over the 50-year time horizon , total costs in the delayed - treatment arm were higher than those in the early treatment arm ( difference of approximately 344,000 sek per patient ) . in summary , the markov model - based estimation revealed that early interferon beta-1b treatment may decrease total costs and increase qalys . however , sensitivity analyses revealed that costs per qaly gained were very high for short time horizons . in fact , only for time horizons longer than 10 years , early treatment was less costly and more effective with respect to qalys gained . this study reached the highest quality of health economic studies ( qhes ) score of the articles reviewed here ( 99 points ) . curkendall et al applied a different approach to assess the pharmacoeconomics of early dmt.62 their analysis was based on insurance claims data in the united states for the years 20002008 , extracted from the thomson reuters marketscan commercial and medicare supplemental databases ; therefore , only health care expenditures and utilizations were analyzed . the core inclusion and exclusion criteria were as follows : ( 1 ) no diagnosis of ms during 12 months before and at the time of a cerebral mri ; this mri scan was defined as start date of the 12-months observation period ; ( 2 ) at least one symptom that was documented 6 months before or 1 month after the index date and that was likely to be attributable to ms ; ( 3 ) initiation of treatment with interferon beta-1a , interferon beta-1b , or ga sometime during the follow - up period . thus , cis patients who may have received dmt after the follow - up period were not included . patients were categorized into two groups an early dmt and a delayed dmt group depending on whether they received dmt before or after the diagnosis of ms . ms diagnosis was defined as one inpatient claim or two outpatients claims coded with icd-9-cm diagnosis code 340 . three thousand nine hundred fifty one patients were enrolled ; 227 and 3,724 were assigned to the early and delayed - treatment cohort , respectively . the mean follow - up was approximately 3 years in both groups , yet the pharmacoeconomic analysis was restricted to the first year after the index mri . the mean time interval between index mri and initiation of dmt was , as expected , significantly shorter in the early treatment group than in the delayed - treatment group ( 122 days versus 184 days ) . however , this difference was small , and more than 90% of the patients assigned to the delayed - treatment group received dmt within the first year after index mri . within the observation period , the early dmt cohort had significantly fewer hospitalizations compared with the delayed dmt cohort ( 0.20 versus 0.33 ) . total drug expenditures were higher in the early dmt cohort ; whereas , total ms - related expenditures were significantly lower in the early dmt group , compared with the delayed dmt group after taking out costs for dmt . both all - cause and ms - related total financial expenditures were not significantly different between the two groups ( all - cause total expenditures us$31,184 in the early dmt group , us$30,051 in the delayed dmt group ) . the authors conclude that the high drug costs of early dmt might be compensated by savings in other medical expenditures . however , the observation period was short , and the analysis was conducted from a third - party payer s perspective alone . the qhes quality score could not be applied to this study , as it does not report on outcome measures , thus not fulfilling the criteria of pharmacoeconomic analyses . in contrast to the other articles reviewed here , the strength of this study lies in the analysis of real costs , albeit restricted to health care expenditures . lazzaro et al applied an open cohort epidemiological model from an italian perspective.63 two treatment arms were analyzed . one group of patients received interferon beta-1b after diagnosis of cis ; the other group was treated with interferon beta-1b after diagnosis of cdms . observation period lasted 25 years with 2,000 cis patients being added to each treatment arm every year , resulting in two cohorts of 50,000 patients each . the conversion rates for years 12 were based on the benefit data,40 while the conversion rates for the remaining 22 years were assumed to decline asymptotically . nineteen years after cdms diagnosis , approximately 50% of the patients would progress to secondary progressive ms . moreover , the observation period was arbitrarily divided into four periods ( year 0 ; years 110 ; years 1120 ; and years 2124 ) . based on the report by kobelt et al,67 edss scores were converted into utility parameters , which were assumed to stay constant within each period . costs were adopted from both the italian national health service ( inhs ) and the societal viewpoints . most of the cost estimates were based on the italian cost - of - illness study by amato et al;68 all costs were reported in 2006 euro . according to this analysis , patients gained 7.84 and 7.49 qalys in the early and delayed treatment arm , respectively ; the difference of 0.35 qalys reached the level of significance . the cua showed that , from the inhs perspective ( ie , only health care resources were considered ) , the icur for early versus delayed interferon treatment was 2,575 per qualy gained and lay considerably below a recently proposed italian willingness to pay of 12,00060,000 per qaly.69 when patient and family resources ( ie , the societal perspective ) were additionally taken into account , early treatment would be even less costly than delayed treatment ( 220,416 versus 226,022 per patient for early versus delayed treatment , respectively ) . the authors conclude that , from the health service and societal perspective , early treatment with interferon beta-1b may be cost - effective , compared with treatment initiation after cdms . however , this interpretation did not hold true for short time horizons as there was a sharp decline in icur estimates over the first years ; eg , from the societal perspective , the icur started from approximately 135,000 per qualy gained at year 1 , decreased below 60,000 after year 3 , and , finally , became negative after year 6 onward . the qhes score of this study reached 75 points and lay considerably below the quality score of the swedish study reviewed here . iskedjian et al performed a pharmacoeconomic analysis of treatment with interferon beta-1a administered intramuscularly once weekly in cis patients ; the study was conducted from a canadian perspective.64 the aim of this study was to investigate : ( 1 ) cost - effectiveness of interferon beta-1a treatment based on the gain of additional monosymptomatic life - years ( mlys ) ; and ( 2 ) cost - utility of long - term interferon beta treatment based on the gain of quality - adjusted monosymptomatic life - years ( qamlys ) . monosymptomatic life - years were defined as years after diagnosis of cis and before diagnosis of cdms . for cis patients , high - dose intravenous steroid pulse therapy ( plus tapering ) for the index event ( current treatment ) and high dose intravenous steroid pulse therapy ( plus tapering ) for the index event plus interferon beta-1a . once the diagnosis of cdms was established , all patients received the same treatment regime with interferon and high dose steroids for treatment of relapse . the time horizon of the cea and cua was 12 and 15 years , respectively , starting from diagnosis of cis and extending to various edss stages of cdms . for the cea and cua , two markov models were developed : the state of cis and various edss states defined the markov cycles ( cycle length , 1 year ) . the conversion rate to cdms was derived from the efficacy results of the controlled high risk avonex multiple sclerosis study ( champs ) study.38 transition probabilities through the various stages of edss were derived from a canadian ms study by weinshenker et al.7 cost analyses , including unemployment rates and the average hospital length of stay within each edss level , were based on data from the literature7072 and canadian health authorities . costs were reported in 2002 canadian dollars ( ca$ ) ; total costs were estimated from the perspective of the canadian ministry of health ( moh ) and from the canadian societal perspective . the former perspective was limited to direct medical costs ; the latter additionally covered direct nonmedical and indirect costs . the input parameters tested in the sensitivity analyses comprised the progression rate to cdms , the indirect costs associated with the duration from cis diagnosis to cdms , the time horizon , the discount rate , and the utilities . the cea demonstrates that the incremental cost - effectiveness ratio ( icer ) of interferon beta per mly gained was ca$53,110 and ca$44,789 from the moh and societal perspectives , respectively . as opposed to the moh perspective , interferon beta treatment was considered cost - effective from the societal perspective , as costs of current treatment per mly gained were higher ( ca$75,444 ) than the icer of interferon beta treatment . the cua was conducted , based on the utility estimates derived from both a canadian and a swedish study.30,70 from the moh perspective , incremental cost of interferon beta per qamly gained ( icur ) was ca$227,586 ( ca$116,071 for the swedish utility estimates ) ; whereas , from the societal perspective , incremental cost of interferon beta reached ca$189,286 ( ca$91,228 , swedish utility estimates ) . the sensitivity analyses showed that both the cea and cua were sensitive to variations of the time horizon with shorter horizons producing higher icer / ucer and vice versa . the multivariate sensitivity analysis of the cea showed that in 87% and 6% of the resulting scenarios the icer of interferon beta therapy would be lower than the costs of current treatment per mly gained from the societal and moh perspective , respectively . therefore , the authors conclude that interferon beta treatment in cis may be cost - effective . however , this conclusion could be drawn from the cea of the societal perspective , only . moreover , concerning the results of the cost - utility analysis , the authors omit to address the issue of decision makers willingness to pay . of the three pharmacoeconomic studies reviewed here , ms is a chronic disease with high economic burden on patients , families , the health system , and society . worldwide incidence and prevalence seems to be increasing with a total of about 2.5 million individuals currently being affected by ms . as reviewed here , several studies have demonstrated that progression of disease severity is accompanied by a rise of total costs as well as a change in the distribution of costs . during early stages of the disease , direct costs , which are mainly caused by dmt , dominate the total costs ; whereas , with accumulation of disability during later stages of the disease especially indirect costs significantly increase.73 moreover , occurrence of relapses has been associated with a peak of costs . noteworthy , quality of life is substantially reduced with increase of disability and during relapse . due to a better understanding of ms pathogenesis and the experience that efficacy of available dmt is high in early stages of ms but declines in the progressive phase of the disease , the concept of early ms treatment has been established . therefore , dmts are usually initiated after diagnosis of relapsing remitting ms or even before definite diagnosis of ms is made at the stage of cis . since dmts are , in general , considered as rather expensive treatments , there has been much debate whether it is economically justifiable to widely prescribe these drugs . a major concern is that clinical trials for approval of dmt even though they have clearly demonstrated efficacy on parameters such as relapses , mri activity , and sometimes also on edss progression during the trial have hardly addressed long - term disability . numerous studies have investigated the pharmacoeconomics of dmt in ms , though only few have explicitly analyzed the pharmacoeconomic effect of early initiation of dmt in cis.6164 these studies indicate that early versus delayed treatment with interferon beta may be overall cost - effective in the long term . reduction of relapses , hospitalization , and indirect costs and a gain of qalys seem to outweigh the costs of dmts . however , to keep in mind , all these analyses and models highly depend on estimates of the applied input parameters . for instance , becker et al74 recently demonstrated the impact of cohort selection by replicating a previously published model - based analysis75 on the cost - effectiveness of interferon beta in ms . based on a different patient cohort with longer follow - up , costs per relapse avoided turned out to be approximately 45% lower than in the original model . in their study reviewed here , iskedjian et al showed that , from the canadian societal perspective , estimated incremental costs of early interferon beta treatment would be 189,286 cad per qamly gained . when applying different utility estimates , the same analysis resulted in 91,228 cad , which is less than 50% of the former result.64 however , it seems to be ambiguous which of the applied utility estimates may be more valid than the other . thus , results of pharmacoeconomic studies and inferences that may be drawn by decision makers largely depend on the analysis approach , the applied simulations and estimations , the perspective of the analysis , and particularly on the quality of the pharmacoeconomic analysis . as shown in our review and for instance in the recent work by yamamoto et al , the latter has turned out to be considerably variable.66 we believe that this is especially critical in a chronic , heterogeneous , and complex disease , such as ms . in this context , one should note , that several pharmacoeconomic studies in ms , including the four studies reviewed here , have been funded by manufacturers of dmt ( table 1 ) . obviously , the results of all four reviewed studies can not be readily generalized to other national settings as several key input data ( eg , costs ) were country - specific.76 besides interferon beta and ga , new dmts have been introduced , which are used as baseline and/or escalation therapy . the wider range of dmts , as well as escalation strategies , needs to be taken into account in future pharmacoeconomic studies , which will be even more challenging . therefore , we need more data on long - term efficacy of dmt and costs in ms in the real - life setting . biomarkers , which will help to stratify patients at early stages with respect to severity of the disease course and response to therapy , are of great need from an economic point of view and even more so for patient care .

backgroundmultidetector computed tomography ( mdct ) angiography represents the standard of reference in the follow - up of patients after endovascular abdominal aortic aneurysm repair ( evar ) , being effective in the detection of the full spectrum of possible complications on both axial and 3d images.methodsthe purpose of this article is to review the normal ct angiography findings of the different types of stent - grafts and to describe the radiological findings of early and late complications after evar on axial and reconstructed images . a selection of cases of post - evar mdct angiography is presented to learn the techniques most commonly used for endovascular treatment , the correct ct scanning technique to acquire the data , the full gamut of possible procedure - related complications and how these complications usually appear on ct images.conclusionmdct angiography is an effective and specific technique in both the pre- and postoperative settings of evar procedures . a better understanding of the procedure , the devices , the normal postoperative imaging features and the possible procedure - related complications ensures optimal planning and follow - up of patients undergoing an evar procedure . multiple sclerosis ( ms ) is a common neurological disease with increasing incidence and prevalence . onset of disease is most frequently in young adulthood when productivity is usually highest ; it is of chronic nature and , in the majority of patients , it will result in accumulation of disability . due to loss of productivity in patients and caregivers as well as high expenses for medical treatment , ms is considered a disease with high economic burden for patients and society . several drugs have been approved for treatment of ms . while treatment ameliorates the course of the disease , it is very costly ; therefore , pharmacoeconomics , evaluating costs and effects of disease - modifying treatment in ms , has become an important issue . here , we review the economic impact and treatment strategies of ms and discuss recent studies on pharmacoeconomics of early treatment with interferon beta .

the head and neck regions are common sites where intra - dermal or subcutaneous firm nodules of 0.5 - 3 cm are found . although its histological findings are well - described , the radiological features of chondroid syringoma have been reported in only few cases . a 59-year - old man presented with a painless mass on the anterior aspect of the upper thigh with a slow enlargement over the past 5 years [ figure 1 ] . dermatological examination showed a relatively firm , mobile 5 3 cm nodule covered by normal skin . laboratory studies including complete blood count , erythrocyte sedimentation rate ( esr ) , urine analysis , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , blood urea , creatinine were normal . sonographic examination revealed mixed , echogenic solid mass measuring approximately 4.5 3 2 cm . on doppler ultrasound , magnetic resonance images ( mri ) revealed a 4.7 2.9 2.2 cm , well - circumscribed , lobular mass that showed heterogeneous enhancement predominantly in the peripheral portion of the mass after intravenous contrast administration [ figure 2 ] . diffusion - weighted imaging ( dwi ) showed areas of increased signal intensity that were indicative of a highly cellular tumor [ figure 3 ] . according to the mri findings , we considered benign subcutaneous lipomatous tumors with cystic and solid components such as angiolipoma or fibrolipoma or well - circumscribed low - grade soft tissue liposarcoma without surrounding tissue extension in our differential diagnosis . because the vascularity of the lesion and heterogeneous pattern with contrast enhancement on mri examination , we excluded lipoma . relatively firm , 5 3 cm , lipoma - like mass covered by normal skin post - contrast axial fat - saturated t1-weighted images show well - circumscribed , lobular mass with heterogeneous enhancement diffusion - weighted imaging ( b : 800 ) signs of increased restriction in diffusion , a finding indicative of a highly cellular tumor we decided to perform trucut biopsy . histopathological examination revealed a group of cells , which have eosinophilic cytoplasm and regular small nuclei , exhibited epithelial - like pattern in the myxoid stroma . however , as adipocytes were variable in size and focally showed a lipoblast - like appearance , some tumor areas were reminiscent of a well - differentiated lipoma - like liposarcoma [ figure 4 ] . microscopic examination shows a focal area of lipoblast - like appearance in the myxoid background ( h and e , 10 ) the patient underwent surgical excision of the mass . there were foreign body reactions against the epidermoid cysts within the tumor and surrounding tissue . epidermoid cysts were seen ( h and e , 5 ) epithelial nests and tubuloalveolar structures in a chondromyxoid stroma . the head and neck regions are common sites ; less commonly , axillary region , hand , foot , abdomen , penis , vulva , scrotum can be involved . chondroid syringoma usually presents as a solitary , asymptomatic , intracutaneous or subcutaneous nodule , mainly seen in middle - aged or older males . the size is usually between 0.5 and 3 cm , but it may be very large . most chondroid syringomas are benign with a slow progression over years . in our case , malignant lesions are usually larger than 3 cm , but benign one measuring 10 cm is also reported . histologically , a wide variety of structures may be found in chondroid syringoma of skin . eccrine types have multiple small syringoid elements and are rich in mucin . in the apocrine type , in our case , there was abundant lipomatous stroma that made diagnostic difficulty in radiological imaging and histopathological examination . previous case reports have described the mr imaging findings of benign and malignant chondroid syringomas.[79 ] although the mr imaging appearance of chondroid syringoma is relatively non - specific , mr imaging generally may allow characterization of the origin of tissue components in the tumor and depiction of the extent and relationships of the lesion to adjacent structures . in our case , the signal intensity characteristics of the lesion ( heterogeneous lesion of mixed fat and fluid - like intensity ) , while not specific for chondroid syringoma , allowed for some analysis of the consistency of the tumor . the benign neoplasms of the subcutaneous tissue that may have similar appearances with our case include superficial lipoma and variants of lipoma ( angiolipoma , myolipoma , spindle - cell and pleomorphic lipoma , chondroid lipoma , and hibernoma ) . although subcutaneous in location , superficial lipoma consists entirely of fat , unlike our lesion . variants of lipoma are commonly heterogeneous and include more than one type of tissue , which may be fat , muscle , chondroid matrix , vascular elements , or myxoid tissue with calcification and hemorrhage . due to the presence of vascular channels on doppler examination , the malignant tumors that may bear similar characteristics with mixed tumor of the skin include well - differentiated liposarcoma , myxoid liposarcoma , extraskeletal myxoid chondrosarcoma , myxofibrosarcoma , and malignant peripheral nerve sheath tumors . after tru - cut biopsy , we considered many of the above entities in the differential diagnosis . there were no cytologic atypia , increased mitotic figures , tumor necrosis , or infiltrative margins , thus excluding malignant chondroid syringoma . the mixed fat and fluid - like signal intensity lesion of skin is somewhat of a diagnostic enigma due to its non - specific imaging and histologic presentation . clearly a soft tissue lesion of mixed fat and fluid - like signal intensity , as in this case , while not pathognomonic for chondroid syringoma , allows for a limited differential diagnosis . as this case shows , chondroid syringoma should be included in the differential diagnosis of a slowly growing , nodular heterogeneous mass in the skin or subcutaneous tissue . 1 . diffusion - weighted imaging , which showed signal intensity changes indicative of a highly cellular tumor in this case , is not routinely done in chondroid syringoma . to our knowledge , this is the first case of chondroid syringoma mimicking low - grade liposarcoma reported in the literature . women have relatively weak neck muscles , which can be a primary cause of chronic fatigue syndrome related to the muscular system , resulting in higher risk of chronic neck pain1 . in relation to pain a study by fisher found that 62% of women wear heels that are over 5 cm , and the ones who enjoy wearing high heels are reported to suffer from foot , knee , and back pain , as well as an alteration of the normal gait patterns2 . according to previous studies , wearing high - heeled footwear can alter the static posture and dynamic movements of the body , and is regarded as a cause of musculoskeletal problems related to the spine3,4,5 . walking in high - heeled shoes changes the kinetic characteristics of the lower extremity joints , causing a reduction in ankle plantar flexor muscle moment , and power during stance phase and increases in forefoot peak pressure . walking in high heels produces an upward displacement of the center of mass of the body and possibly a more unstable posture compared to that producted by low - heeled walking6,7,8,9 . additionally , shock and ground reaction forces can lead to increased axial pressure onto the intervertebral discs , resulting in increased erector spinal muscle activation10 . walking in high - heeled shoes produce an increase in the ground reaction force compared to flat heeled shoes . in theory , the increase in ground reaction force with increased height may result from decreased subtalar joint pronation at heel strike and a lengthened tibiofemoral lever arm . as a result , trunk muscle activity increases with increasing heel height , which changes the posture and ground reaction force , leading to discomfort of the back muscle in women who wear high heels11 . in addition , the effects are magnified with increased activation of the erector spinae muscles , which contribute to compression of the spine . therefore , wearing high heels for an extended period can lead to an increase in paraspinal musculature activation of the lumbar and cervical spine , causing prolonged overload and fatigue in the trunk and neck muscles1 . a recent study reported that walking in heels induced a significant increase in the activation of cervical muscles in healthy subjects . this study was conducted in order to examine the effects of wearing high heels during gait on the cervical and lumbar musculature in patients with chronic neck pain and in healthy women . thirteen healthy women were recruited for this study . an information leaflet describing the study method and procedure was used for the recruitment of subjects . the inclusion criteria for patients were as follows : ( 1 ) history of neck pain for the past three months or less , ( 2 ) between the age of 2045 years , ( 3 ) not experiencing orthopedic or neurological problems other than neck pain , and ( 4 ) able to wear high heels without pain . this study excluded subjects who felt acute worsening of pain symptoms during their maximal effort or who were in an unstable condition with an acute flare up during the time of testing . table 1table 1.demographic data of the collective intention to treat ( n=13)variablemeanstandard deviationage ( yrs)31.56.6height ( cm)161.14.2body mass ( kg)55.17.5 shows a list of common characteristic of the participants in this study . a test on the general characteristics of the subjects was filled out prior to conduct of this study . to determine the effects of the different heights of heels on the muscle activation , the paraspinalis cervicis ( cervical spine ) and erector spinae ( lumbar spine ) were measured at the time of heel strike and toe off during gait . after preparing the skin by shaving , cleaning with alcohol , and sanding , two differential surface electrodes were placed at a distance of 2 cm from each other . surface electromyography ( emg ) electrodes were placed on the paraspinalis cervicis and erector spinae of the subjects dominant side . all electrodes were placed over the muscle mass , which was visible during maximally resisted voluntary contraction . two foot switches were placed and attached on the shoe exterior , underneath the first metatarsal head area and the heel of the shoe on the dominant side . to familiarize the subjects to the testing condition with surface electromyography ( emg ) attached to their body , they were instructed to walk down the walkway three times prior to the testing condition . each subject was instructed to walk naturally at a self - selected velocity on a flat and even surface under three different testing conditions ( barefoot , 4 cm stiletto heels , and 10 cm stiletto heels with a base of 1 cm ) . prior to each gait trial , subjects were instructed to stand still with their feet together in a comfortable position , and then were asked to look straight up and walk down the 10 m walkway in front of them . to prevent muscular fatigue , emg data collected during the 10 m walk , the data during the middle 6 m block was used for analysis . data from the three trials were averaged for analysis using the heel strike and toe off events for each gait trial . all subjects with neck pain were in a stable condition without an acute flare up during the time of testing . differences in the emg activity of the paraspinalis cervicis and erector spinae in patients with neck pain during heel strike and toe off during gait were assessed using the spss 12.0 program ( spss inc . , a one - way repeated anova was used for the identification of significant differences among the barefoot , 4 cm high heel , and 10 cm high heel conditions ; and a post - hoc test using the least - significant - difference ( lsd ) method was employed for comparing the conditions . muscle activation in the paraspinalis cervicis and erector spinae at heel strike and toe off ( except that of paraspinalis cervicis at toe off event ) differed significantly with the 10 cm high heels , compared to the barefoot condition . at heel strike , muscle activation of the paraspinalis cervicis was significantly greater with the 10 cm high heels as compared to that with the 4 cm high heels ( table 2table 2.muscle activation ( % mvic ) in normal subjects under the three conditions ( n = 13)musclesgait cyclebarefoot4 cm high - heeled10 cm high - heeledcervical paraspinaeheel strike7.62.17.72.49.43.2toe off7.51.87.11.68.22.5erector spinaeheel strike24.78.229.510.531.212.3toe off28.310.530.311.136.218.3meansd . % mvic : % maximum voluntary isometric contraction . * significant differences compared to barefoot condition . % mvic : % maximum voluntary isometric contraction . * significant differences compared to barefoot condition . this study demonstrated the effects of walking in high heels on the muscular activity of the cervical and lumbar muscles in healthy women . previous studies have reported the muscle activation or biomechanical effects of walking in high heels in healthy women . mika and coworkers studied the effects of walking on barefoot , 4 cm high heels , and 10 cm high heels on the activation of the cervical paraspinalis in young women ( 2025 years ) and older women ( 4555 years ) . the results showed a significant increase in cervical muscle activation while walking in 10 cm high heels . subjects in the older group showed a significant increase in the cervical muscle activation at heel strike while walking in 4 cm high heels and 10 cm high heels . a higher magnitude of vertical ground reaction force was demonstrated with high heels , and the effects were amplified with the presence of more active erector spinae muscles , which could act partially to compress the spine12 , 13 . results of previous studies have demonstrated an increase in the compensated muscular activities with an increasing heel height . wearing high heels changes the overall body posture and loading on the spine and the joints in the lower extremities , resulting in a change in the body s center of mass ( com ) . snow and williams reported that wearing excessively high heels induces a shift in the body s com anteriorly and superiorly . ankle is shifted superiorly and anteriorly , and the base of support ( bos ) shifts anteriorly . as the body tries to maintain this posture without falling , compensatory activity of both the erector spinae and the cervical paraspinalis increases14 . cervical spine movement assists in maintaining the head stability in space , assists in dynamic postural control , and compensates for trunk motion to maintain head stability during gait . with neck extension , the center of mass of the head neck extension may also assist in balancing the head over the trunk due to the center of mass of the head lying anterior to the cervical spine . previous studies also attempted to determine the lumbar curve angle , center of mass , and activation of the erector spinae during the gait of healthy women wearing high - heeled shoes6 , 15 . the results indicated a significant decrease in the lumbar curve angle and , in the vertical movement of the body s center of mass , and a significant increase in activation of the erector spinae . our results showed a significant increase in muscle activation of the erector spinae at heel strike and toe - off events while wearing 10 cm high heel , as compared to that in barefoot condition in all subjects . the results of our study demonstrated that wearing high - heeled shoes increases the activation of cervical and lumbar musculature in healthy women16 . according to mika and colleagues , even healthy people wearing high - heeled shoes experience increased back muscle activity during gait , which could promote local muscle fatigue , in turn leading to tissue deformation , such as swelling or decreased movement13 . in addition , these symptoms may be more remarkable while wearing stiletto type high heels with unstable balance . further studies are needed to evaluate the long - term effects between high - heeled shoes and the mechanism of muscle activation of the cervical and lumbar spine . another limitation of this study was the small number of recruited subjects . for a more accurate observation of the relationship between neck pain and high - heeled shoes , a follow - up study with a longer period of time and future studies will be necessary to evaluate the effect of high - heels on cervical spine kinematics and muscle fatigue over an extended period of time . in addition , consideration of changes in body alignment and muscle activation caused by extrinsic factors , such as high - heeled shoes , may be a more effective approach for planning treatment strategies .

chondroid syringoma , or mixed tumor of skin , is a relatively rare , usually benign sweat gland tumor , most often seen in the head - and - neck region . rare malignant examples have been reported , commonly involving the extremities . we report here a case radiologically mimicking a malignant neoplasm , but histologically - proven benign subcutaneous chondroid syringoma , arising in the anterior aspect of the upper thigh of a 59-year - old male . [ purpose ] the purpose of this study was to investigate the effects of different height of high heels on muscle activation of the paraspinalis cervicis and erector spinae in healthy young women . [ subjects and methods ] thirteen healthy women were recruited in this study . to examine the effects of different heights of heels on muscle activation , the paraspinalis cervicis ( cervical spine ) and erector spinae ( lumbar spine ) were measured at the time of heel strike and toe off during gait on three different conditions ( barefoot , 4 cm high heels , and 10 cm high heels ) . there are no previous trials or reports that have evaluated this approach in patients with chronic neck pain . [ results ] a significant increase in muscle activation of the paraspinalis cervicis and erector spinae at heel strike and toe off ( except that of the paraspinalis cervicis at toe off in healthy subjects ) was observed in the under 10 cm high heel condition as , compared to that with barefoot condition , in all the subjects . [ conclusion ] the height of the high heels affects to the activation demand of the paraspinalis cervicis and erector spinae in patients with neck pain .

life expectancy is among the objective indexes for measuring health for a person , society , or population . in 1880 - 1882 , life expectancy at birth for females in jamaica was 39.8 years which was 2.79 years more than that for males . one hundred and twenty - two year ( 2002 - 2004 ) , this health disparity increased to 5.81 years : life expectancy at birth for female was 77.07 years . for the world , the difference in life expectancy for the sexes was 4.2 years more for females than males : for 2000 - 2005 , life expectancy at birth for females was 68.1 years . within the expanded conceptual framework offered by the world health organization ( who ) in the late 1940s , health is more than the absence of morbidity as it includes social , psychological and physiological wellbeing . some scholars opined that using the opposite of ill - health to measure health is a negative approach is health is more than this biomedical approach . brannon and feist forwarded a positive approach which is in keeping with the biopsychosocial framework developed by engel . engel coined the term biopsychosocial when he forwarded the perspective that patient care must integrate the mind , body and social environment[58 ] . he believed that mentally patient care is not merely about the illness , as other factors are equally influence the health of the patient . although this was not new because the who had already stated this , it was the application which was different from the traditional biomedical approach to the study and treatment of ill patients . embedded in engel 's works were wellbeing , wellness and quality of life and not merely the removal of the illness , which psychologists like brannon and feist called the positive approach to the study and treatment of health . recognizing the limitation of life expectancy , who therefore developed dale disability adjusted life expectancy which discounted life expectancy by number of years spent in illness . the emphasis in the 21 century therefore was healthy life and not length of life ( i.e. life expectancy ) . dale is the years in ill health which is weighted according to severity , which is then subtracted from the expected overall life expectancy to give the equivalent healthy years of life . using healthy years , statistics revealed that the health disparity between the sexes in jamaica was 5 years in 2007 , indicating that health status of females on average in jamaica is better than that for males . this is not atypical to jamaica as females in many nations had a greater healthy life expectancy than males . the discipline of public health is concerned with more than accepting the health disparity as indicated by life expectancy or healthy life expectancy , as it seeks to improve the quality of life of the populace and the various subgroups that are within a particular geographical border . in order for this mandate to be attained , we can not exclude the study of females health merely because they are living longer than males and accept this as a given ; and that there is not need therefore to examine their health status . many empirical studies that have examined health of caribbean nationals were on the population[1115 ] ; elderly[1625 ] ; children ; adolescents[2830 ] and females have been omitted from the discourse . the values for the healthy life expectancy can not be enough to indicator the health status of females neither can use health status of population , children , elderly and adolescents to measure that of females . who forwarded a position that there is a disparity between contracting many diseases and the gender constitution of an individual , suggesting that population health can not be used to measure female health . females have a high propensity than males to contract particular conditions such as depression , osteoporosis and osteoarthritis . a study conducted by mcdonough and walters revealed that women had a 23 percent higher distress score than men and were more likely to report chronic diseases compared to males ( 30% ) . it was found that men believed their health was better ( 2% higher ) than that self - reported by females . mcdonough et al used data from a longitudinal study named canadian national population health survey ( nphs ) . the current study fills the gap in the health literature by investigating health of females in jamaica . the objectives of the current study are 1 ) to examine the health conditions ; 2 ) provide an epidemiological profile of changing health conditions in the last one half decade ( 2002 - 2007 ) ; 3 ) evaluate whether self - reported illness is a good measure of health status ; 4 ) compute the mean age of females having particular health conditions ; 5 ) calculate the mean age of being ill compared with those who are not ill ; and 6 ) assess the correlation between health status and income quintile . the current study extracted subsample of females from two secondary cross - sectional data collected by the planning institute of jamaica and the statistical institute of jamaica . in 2002 , a subsample of 12,675 females was extracted from the sample of 25,018 respondents and for 2007 ; a subsample of 3,479 females was extracted from 6,783 respondents . the survey is called the jamaica survey of living conditions ( jslc ) which began in 1989 . the jslc is modification of the world bank 's living standards measurement study ( lsms ) household survey . trained data collectors are used to gather the data ; and these individuals are trained by the statistical institute of jamaica the survey was drawn using stratified random sampling . this design was a two - stage stratified random sampling design where there was a primary sampling unit ( psu ) and a selection of dwellings from the primary units . the psu is an enumeration district ( ed ) , which constitutes a minimum of 100 residences in rural areas and 150 in urban areas . this means that the country was grouped into strata of equal size based on dwellings ( eds ) . based on the psus , a listing of all the dwellings was made , and this became the sampling frame from which a master sample of dwelling was compiled , which in turn provided the sampling frame for the labor force . one third of the labor force survey ( i.e. lfs ) was selected for the jslc . self - reported illness ( or health conditions ) : the question was asked : is this a diagnosed recurring illness ? the answering options are : yes , cold ; yes , diarrhea ; yes , asthma ; yes , diabetes ; yes , hypertension ; yes , arthritis ; yes , other ; and no . self - rated health status ( health status ) : how is your health in general ? and the options were very good ; good ; fair ; poor and very poor . the first time this was collected for jamaicans , using the jslc , was in 2007 . social class : this variable was measured based on the income quintiles : the upper classes were those in the wealthy quintiles ( quintiles 4 and 5 ) ; middle class was quintile 3 and poor those in lower quintiles ( quintiles 1 and 2 ) . health care - seeking behavior : this is a dichotomous variable which came from the question has a doctor , nurse , pharmacist , midwife , healer or any other health practitioner been visited ? with the option ( yes or no ) . the data were collected , stored and retrieved in spss for windows 16.0 ( spss inc ; chicago , il , usa ) . descriptive statistics were used to provide information on the socio - demographic variables of the sample . cross tabulations were employed to examine correlations between non - metric variables , and analysis of variance ( anova ) was utilized to examine statistical associations between a metric and non - metric variable . the level of significance used in this research was 5% ( i.e. 95% confidence interval ) . bryman and cramer correlation coefficient values were used to determine , the strength of a relation between ( or among ) variables : 0.19 and below , very low ; 0.20 to 0.39 , low ; 0.40 to 0.69 , moderate ; 0.70 to 0.89 , high ( strong ) ; and 0.90 to 1 is very high ( very strong ) . the data were collected , stored and retrieved in spss for windows 16.0 ( spss inc ; chicago , il , usa ) . descriptive statistics were used to provide information on the socio - demographic variables of the sample . cross tabulations were employed to examine correlations between non - metric variables , and analysis of variance ( anova ) was utilized to examine statistical associations between a metric and non - metric variable . the level of significance used in this research was 5% ( i.e. 95% confidence interval ) . bryman and cramer correlation coefficient values were used to determine , the strength of a relation between ( or among ) variables : 0.19 and below , very low ; 0.20 to 0.39 , low ; 0.40 to 0.69 , moderate ; 0.70 to 0.89 , high ( strong ) ; and 0.90 to 1 is very high ( very strong ) . the data were collected , stored and retrieved in spss for windows 16.0 ( spss inc ; chicago , il , usa ) . descriptive statistics were used to provide information on the socio - demographic variables of the sample . cross tabulations were employed to examine correlations between non - metric variables , and analysis of variance ( anova ) was utilized to examine statistical associations between a metric and non - metric variable . the level of significance used in this research was 5% ( i.e. 95% confidence interval ) . bryman and cramer correlation coefficient values were used to determine , the strength of a relation between ( or among ) variables : 0.19 and below , very low ; 0.20 to 0.39 , low ; 0.40 to 0.69 , moderate ; 0.70 to 0.89 , high ( strong ) ; and 0.90 to 1 is very high ( very strong ) . in 2002 , 14.7% of sample reported an illness and this increased by 19.1% in 2007 . over the same period , health insurance coverage increased by 81.0% ( to 21.0% in 2007 ) ; those seeking medical care increased to 67.6% ( from 66.0% ) ; the mean age in 2007 was 30.621.9 years which marginal increased from 29.4 22.3 years ; diabetic cases exponentially increased by 227.7% ( in 2007 , 15.4% ) ; hypertension decline by 45.5% ( to 24.8% in 2007 ) and arthritic cases felt by 66.1% ( to 9.4% in 2007 ) . urbanization was evident between 2007 and 2002 as the number of females who resided in urban areas increased by 114.7% ( to 30.4% in 2007 ) , with a corresponding decline of 19.4% in females zones . table 1 revealed that the increase in self - reported illness was substantially accounted for by increased cases in the rural sample ( from 12.9% in 2002 to 20.0% in 2007 ) . the drastic increase in health insurance coverage in 2007 was due to public establishment of public health insurance coverage . the greatest increase was observed in semi - urban areas 17.8% ) followed by urban ( 9.6% ) and rural ( 7.8% ) ( table 1 ) . the increases in self - reported illness can be accounted for by diabetes mellitus , asthma and other dysfunctions . concurrently , most of the increased cases were diabetic in semi - urban zones ( 17.1% ) ; other health conditions in semi - urban areas ( 12.4% ) and asthma in urban zones ( 12.0% ) ( table 1 ) . sociodemographic characteristics of sample by area of residence , 2002 and 2007 there was a significant statistical correlation between health status and self - reported illness - ( df = 4 ) = 700.633 , p < 0.001 ; with there being a negative moderate relation between the variables correlation coefficient = - 0.412 ( table 2 ) . based on table 2 , 10.7% of those who reported an illness had had very good health status compared to 40.2% of those who did not indicate an illness . on the other hand , 2.5% of those who did not report a dysfunction had at least poor health status compared to 19.8% of those who indicated having an illness . even after controlling health status and self - reported illness by age , marital status and per capita annual expenditure , a moderate negative correlation was found - correlation coefficient = - 0.362 . health status by self - reported illness , 2007 on further examination of the self - reported illness by age , it was found that in 2002 the mean age of individual who reported an illness was 43.97 26.81 years compared to 27.05 20.41 years for who without an illness t - test = 30.818 , p < 0.001 . in 2007 , the mean age of reporting an illness was 42.83 26.53 years compared to 28.16 19.95 years for those who did not report an ailment t - test = 15.263 , p < 0.001 . based on figure 1 , there is an increase in the mean age of females being diagnosed with diarrhea ( 32.00 36.2 years ) and asthma ( 21.73 20.51 years ) . however , there is reduction in the mean age of females reported being diagnosed with chronic illness such as diabetes mellitus ( 60.54 17.14 years ) ; hypertension ( 60.85 16.93 years ) and arthritis 59.72 15.41 years ) . the greatest decline in mean age of chronically ill diagnosed females was in arthritic cases ( by 7.41 years ) . concurrently , the mean age of females with unspecified health conditions fell by ( 33% , from 54.62 21.77 years in 2002 to 36.42 23.69 years in 2007 ) . a cross tabulation between health status and income quintile revealed a significant statistical correlation - ( df = 16 ) = 54.044 , p < 0.001 ; with the relationship being a very weak one - correlation coefficient = 0.126 ( table 3 ) . based on table 3 , the wealthy reported the greatest health status ( i.e. very good ) compared to the wealthiest 20% ( 36.7% ) ; with the poorest 20% recorded the least very good health status . health status by income quintile , 2007 no significant statistical correlation was found between diagnosed self - reported illness and income quintile - ( df = 28 ) = 36.161 , p > 0.001 ( table 4 ) . in 2002 , 14.7% of sample reported an illness and this increased by 19.1% in 2007 . over the same period , health insurance coverage increased by 81.0% ( to 21.0% in 2007 ) ; those seeking medical care increased to 67.6% ( from 66.0% ) ; the mean age in 2007 was 30.621.9 years which marginal increased from 29.4 22.3 years ; diabetic cases exponentially increased by 227.7% ( in 2007 , 15.4% ) ; hypertension decline by 45.5% ( to 24.8% in 2007 ) and arthritic cases felt by 66.1% ( to 9.4% in 2007 ) . urbanization was evident between 2007 and 2002 as the number of females who resided in urban areas increased by 114.7% ( to 30.4% in 2007 ) , with a corresponding decline of 19.4% in females zones . table 1 revealed that the increase in self - reported illness was substantially accounted for by increased cases in the rural sample ( from 12.9% in 2002 to 20.0% in 2007 ) . the drastic increase in health insurance coverage in 2007 was due to public establishment of public health insurance coverage . the greatest increase was observed in semi - urban areas 17.8% ) followed by urban ( 9.6% ) and rural ( 7.8% ) ( table 1 ) . the increases in self - reported illness can be accounted for by diabetes mellitus , asthma and other dysfunctions . concurrently , most of the increased cases were diabetic in semi - urban zones ( 17.1% ) ; other health conditions in semi - urban areas ( 12.4% ) and asthma in urban zones ( 12.0% ) ( table 1 ) . sociodemographic characteristics of sample by area of residence , 2002 and 2007 there was a significant statistical correlation between health status and self - reported illness - ( df = 4 ) = 700.633 , p < 0.001 ; with there being a negative moderate relation between the variables correlation coefficient = - 0.412 ( table 2 ) . based on table 2 , 10.7% of those who reported an illness had had very good health status compared to 40.2% of those who did not indicate an illness . on the other hand , 2.5% of those who did not report a dysfunction had at least poor health status compared to 19.8% of those who indicated having an illness . even after controlling health status and self - reported illness by age , marital status and per capita annual expenditure , a moderate negative correlation was found - correlation coefficient = - 0.362 . health status by self - reported illness , 2007 on further examination of the self - reported illness by age , it was found that in 2002 the mean age of individual who reported an illness was 43.97 26.81 years compared to 27.05 20.41 years for who without an illness t - test = 30.818 , p < 0.001 . in 2007 , the mean age of reporting an illness was 42.83 26.53 years compared to 28.16 19.95 years for those who did not report an ailment t - test = 15.263 , p < 0.001 . based on figure 1 , there is an increase in the mean age of females being diagnosed with diarrhea ( 32.00 36.2 years ) and asthma ( 21.73 20.51 years ) . however , there is reduction in the mean age of females reported being diagnosed with chronic illness such as diabetes mellitus ( 60.54 17.14 years ) ; hypertension ( 60.85 16.93 years ) and arthritis 59.72 15.41 years ) . the greatest decline in mean age of chronically ill diagnosed females was in arthritic cases ( by 7.41 years ) . concurrently , the mean age of females with unspecified health conditions fell by ( 33% , from 54.62 21.77 years in 2002 to 36.42 23.69 years in 2007 ) . a cross tabulation between health status and income quintile revealed a significant statistical correlation - ( df = 16 ) = 54.044 , p < 0.001 ; with the relationship being a very weak one - correlation coefficient = 0.126 ( table 3 ) . based on table 3 , the wealthy reported the greatest health status ( i.e. very good ) compared to the wealthiest 20% ( 36.7% ) ; with the poorest 20% recorded the least very good health status . health status by income quintile , 2007 no significant statistical correlation was found between diagnosed self - reported illness and income quintile - ( df = 28 ) = 36.161 , p > 0.001 ( table 4 ) . in 2002 , 14.7% of sample reported an illness and this increased by 19.1% in 2007 . over the same period , health insurance coverage increased by 81.0% ( to 21.0% in 2007 ) ; those seeking medical care increased to 67.6% ( from 66.0% ) ; the mean age in 2007 was 30.621.9 years which marginal increased from 29.4 22.3 years ; diabetic cases exponentially increased by 227.7% ( in 2007 , 15.4% ) ; hypertension decline by 45.5% ( to 24.8% in 2007 ) and arthritic cases felt by 66.1% ( to 9.4% in 2007 ) . urbanization was evident between 2007 and 2002 as the number of females who resided in urban areas increased by 114.7% ( to 30.4% in 2007 ) , with a corresponding decline of 19.4% in females zones . table 1 revealed that the increase in self - reported illness was substantially accounted for by increased cases in the rural sample ( from 12.9% in 2002 to 20.0% in 2007 ) . the drastic increase in health insurance coverage in 2007 was due to public establishment of public health insurance coverage . the greatest increase was observed in semi - urban areas 17.8% ) followed by urban ( 9.6% ) and rural ( 7.8% ) ( table 1 ) . the increases in self - reported illness can be accounted for by diabetes mellitus , asthma and other dysfunctions . concurrently , most of the increased cases were diabetic in semi - urban zones ( 17.1% ) ; other health conditions in semi - urban areas ( 12.4% ) and asthma in urban zones ( 12.0% ) ( table 1 ) . there was a significant statistical correlation between health status and self - reported illness - ( df = 4 ) = 700.633 , p < 0.001 ; with there being a negative moderate relation between the variables correlation coefficient = - 0.412 ( table 2 ) . based on table 2 , 10.7% of those who reported an illness had had very good health status compared to 40.2% of those who did not indicate an illness . on the other hand , 2.5% of those who did not report a dysfunction had at least poor health status compared to 19.8% of those who indicated having an illness . even after controlling health status and self - reported illness by age , marital status and per capita annual expenditure , a moderate negative correlation was found - correlation coefficient = - 0.362 . health status by self - reported illness , 2007 on further examination of the self - reported illness by age , it was found that in 2002 the mean age of individual who reported an illness was 43.97 26.81 years compared to 27.05 20.41 years for who without an illness t - test = 30.818 , p < 0.001 . in 2007 , the mean age of reporting an illness was 42.83 26.53 years compared to 28.16 19.95 years for those who did not report an ailment t - test = 15.263 , p < 0.001 . based on figure 1 , there is an increase in the mean age of females being diagnosed with diarrhea ( 32.00 36.2 years ) and asthma ( 21.73 20.51 years ) . however , there is reduction in the mean age of females reported being diagnosed with chronic illness such as diabetes mellitus ( 60.54 17.14 years ) ; hypertension ( 60.85 16.93 years ) and arthritis 59.72 15.41 years ) . the greatest decline in mean age of chronically ill diagnosed females was in arthritic cases ( by 7.41 years ) . concurrently , the mean age of females with unspecified health conditions fell by ( 33% , from 54.62 21.77 years in 2002 to 36.42 23.69 years in 2007 ) . a cross tabulation between health status and income quintile revealed a significant statistical correlation - ( df = 16 ) = 54.044 , p < 0.001 ; with the relationship being a very weak one - correlation coefficient = 0.126 ( table 3 ) . based on table 3 , the wealthy reported the greatest health status ( i.e. very good ) compared to the wealthiest 20% ( 36.7% ) ; with the poorest 20% recorded the least very good health status . health status by income quintile , 2007 no significant statistical correlation was found between diagnosed self - reported illness and income quintile - ( df = 28 ) = 36.161 , p > 0.001 ( table 4 ) . the current study found a moderate significant correlation between the two aforementioned variables , suggesting that self - reported illness is a relatively good measure of female 's health . in this study it was revealed that 60 out of every 100 who reported an illness had at most fair health status , with 20 out every 100 indicated a least poor health . it is evident from the findings that health status is wider than illness , which concurs with the literature , which is keeping with the propositions of the who that health must be more than the absence of illness . health status is people 's self - rated perspective on their general health status , which includes a percentage of poor health ( or ill - health ) . the other components of this status include life satisfaction , happiness , and psychosocial wellbeing . using a sample of elderly barbadians , hambleton et there is a disparity between the current study and that of hambleton 's work as more of health status of the elderly is explained by current illness with this being less for females in jamaica . concomitantly , there is an epidemiological shift in the typology of illnesses affecting females as the change is towards diabetes mellitus . in 2007 over 2002 , the 15 out of every 100 females reported being diagnosed with diabetes mellitus compared to 5 in 100 in 2002 indicating the negative effects of life behavior of female 's health status . another important finding of the current study is that diagnosed illnesses are not significant different based on income quintile in which a female is categorized . however , the health status of females in different social standing ( measured using income quintile ) is different . embedded in this finding is the role of income plays in improving health status . like marmot , this study found that income is able to buy some improvement in health status ; but this work goes further as it found that income it does not reduce the typology is health conditions affecting the female . before this discussion can proceed , the discourse must address the biases in subjective indexes which are found in studies like this one . any study on subjective indexes in the measurement of health ( for example , happiness , life satisfaction ; health status , self - reported illness ) needs to address the challenges of biases that are found in self - reported data in particular self - reported health data . the discourse of subjective wellbeing using survey data can not deny that it is based on the person 's judgment , and must be prone to systematic and non - systematic biases . diener argued that the subjective measure seemed to contain substantial amounts of valid variance , suggesting that there is validity to the use of this approach in the measurement of health ( or wellbeing ) like the objective indexes such as life expectancy , mortality or diagnosed morbidity . a study by finnas et al opined that there are some methodological issues surround the use of self - reported ( or self - rated ) health and that these may result in incorrect inference ; but that this measure is useful in understanding health , morbidity and mortality . using life expectancy and self - reported illness data for jamaicans , bourne found a strong significant correlation between the two variables ( correlation coefficient , r = - 0.731 ) , and that self - reported illness accounted for 54% of the variance in life expectancy . when bourne disaggregated the life expectancy and self - reported illness data by sexes , he found a strong correlation between males ( correlation coefficient , r = 0.796 ) than for females ( correlation coefficient , r = 0.684 ) . self - reported data therefore do have some biases ; but that it is good measure for health in jamaica and more so for males . in spite of this fact , the current research recognized some of the problems in using self - reported health data ( read finnas et al . for more information ) , while providing empirical findings using people 's perception on their health . now that the discourse on objective and subjective indexes of is some out of the way , the next issue of concern is the reduced aged of reported illness and age of being diagnosed with particular chronic illness . in 2002 , the mean age recorded for those who self - reported an illness was 44 years and this fell by 1 year in 2007 , indicating that on average females are becoming diagnosed with an illness 2 months earlier . when self - reported illness was disaggregated into acute and chronic health conditions , it was revealed that on average females were being diagnosed 7.41 years earlier with arthritis in 2007 over 2002 ; 4.95 years earlier with hypertension and 1.13 years earlier with diabetes mellitus . the current study has revealed that , although healthy life expectancy for females at birth in jamaica is 66 years , improvements in their health status can not be neglected as there are shits in health conditions ( to diabetes mellitus ) as well as the decline in ages at which females are being diagnosed with particular chronic illnesses . frontometaphyseal dysplasia ( fmd ) , also called gorlin cohen syndrome , is a hereditary x - linked dominant syndrome described in 1969 with less than 30 cases described in the literature . this case report of a child with fmd is presented owing to the rarity of the syndrome and the anticipated difficult airway , which was successfully managed by using a combination of dexmedetomidine and ketamine while preserving spontaneous ventilation . a 2-year - old female child , a known case of fmd , presented for open reduction of the left hip with osteotomy of femur . physical examination revealed a slender undernourished girl of 8 kg with prominent supraorbital ridges , ocular hypertelorism , low set ears and a wide bridge nose with prominent eyes . airway examination revealed a mallampatti score of iii with significant retrognathia , high arched palate with malocclusion of teeth . in addition , she had dorsolumbar scoliosis , pectus carinatum , bowing of long bones with distal phalangeal hypoplasia and multiple joint dislocations [ figure 1 ] . pre - operative blood investigations , echocardiography and chest x - ray were within physiological limits . on arrival to the operation theatre , monitors were connected and child pre - oxygenated for 5 minutes . injection dexmedetomidine 1 g / kg was administered for 10 min and then a continuous infusion at 1 g / kg / h was set for the duration of the remaining procedure . ketamine was administered in increments of 5 mg up to 12 mg until there was no response to jaw thrust while ensuring spontaneous respiration . just before direct laryngoscopy intravenous lignocaine rigid laryngoscopy with miller 1 straight blade offered a grade iv cormack and lehane view . after optimal external laryngeal manipulation , the visible glottic chink was sprayed with topical lignocaine and tracheal intubation was successfully performed using an uncuffed 4 sized endotracheal tube . anesthesia was continued with n2o in 40% o2 along with a continuous dexmedetomidine and atracurium infusion . adequate padding was provided at pressure points and extreme caution was exercised during positioning . at the end of fmd belongs to the otopalatodigital spectrum syndromes that includes four phenotypically related conditions , otopalatodigital syndrome types 1 and 2 , fmd and melnick - needles syndrome . the most common manifestations include supraorbital hyperostosis , hypertelorism , down - slanting palpebral fissures , broad nasal bridge and micrognathia with anomalies of teeth and generalized skeletal dysplasia . congenital heart disease , subglottic tracheal narrowing and genitourinary anomalies , muscular hypotonia . micrognathia , microstomia and malocclusion of teeth may make direct laryngoscopy impossible ; therefore , a well - planned airway strategy is mandatory . ketamine was preferred in our case of anticipated difficult airway due to it 's inherent sympathomimetic actions devoid of respiratory depression alongwith provision of excellent analgesia and amnesia . dexmedetomidine a specific and selective 2-adrenoceptor agonist known for its sedative , anxiolytic , analgesic properties was used to complement ketamine . at the same time dexmedetomidine offsets the sympathomimetic effects of ketamine , this unique pharmacological combination in the present case preserved the respiratory drive , allowed maintenance of a patent airway and provided sufficient sedation , analgesia and anesthesia to allow successful airway control . in addition , topical lignocaine was used as per recommendation of aroni et al . which states that ketamine does not depress coughing or swallowing reflexes . available literature describes the use of combination of both these drugs in children during procedural anesthesia and not as a complete anesthesia protocol in a challenging case . the present experience of using this combination successfully paves the way to emerging new solutions for management of a difficult pediatric airway . hence safety profile , rapid onset of action with adequate sedation and analgesia provided by the ketamine and dexmedetomidine make them a distinctive drug combination in the pediatric difficult airway situation in a child with fmd .

background : the 21st century can not see the examination of health status of elderly , population , children and adolescents ; but not for females.aims:current study are 1 ) to examine the health conditions ; 2 ) provide an epidemiological profile of changing health conditions in the last one half decade ( 2002 - 2007 ) ; 3 ) evaluate whether self - reported illness is a good measure of health status ; 4 ) compute the mean age of females having particular health conditions ; 5 ) calculate the mean age of being ill compared with those who are not ill ; and 6 ) assess the correlation between health status and income quintile.methods and results : in 2002 , a subsample of 12,675 females was extracted from the sample of 25,018 respondents and for 2007 ; a subsample of 3,479 females was extracted from 6,783 respondents.results:there is reduction in the mean age of females reported being diagnosed with chronic illness such as diabetes mellitus ( 60.54 17.14 years ) ; hypertension ( 60.85 16.93 years ) and arthritis 59.72 15.41 years ) . the greatest decline in mean age of chronically ill diagnosed females was in arthritic cases ( by 7.41 years ) . concurrently , the mean age of females with unspecified health conditions fell by ( 33% , from 54.62 21.77 years in 2002 to 36.42 23.69 years in 2007).conclusion : although healthy life expectancy for females at birth in jamaica was 66 years , improvements in their health status can not be neglected as there are shits in health conditions ( to diabetes mellitus ) as well as the decline in ages at which females are being diagnosed with particular chronic illnesses . frontometaphyseal dysplasia ( fmd ) , also called gorlin - cohen syndrome , is a rare hereditary x - linked dominant craniotubular bone disorder . the presentation describes the airway management of a 2-year - old child suffering from fmd with significant retrognathia , posted for major long bone corrective osteotomy . induction with a combination of dexmedetomidine and ketamine preceded a successful endotracheal intubation under spontaneous ventilation .

multiple sclerosis ( ms ) is a chronic , autoimmune , demyelinating disease of the central nervous system ( cns ) . presenting symptoms can vary greatly , but most commonly involve weakness , paresthesia , gait difficulty , or visual deficits . virtually any area of the cns white matter can be involved , though this most classically involves the periventricular white matter . brain stem involvement is common , though isolated cranial nerve palsies are rare signs in ms . previous studies have suggested that isolated cranial nerve palsies in ms are more commonly found as presenting symptoms than as relapsing symptoms . among isolated cranial nerve palsies in ms , the fifth nerve is most commonly involved ( 4.8% ) , followed by the seventh nerve ( 3.7% ) , and the sixth nerve ( 1.0% ) . thus , abducens palsy is a rare isolated ms finding , either as a presenting sign or during disease exacerbation . patients with abducens palsy typically present with diplopia upon horizontal gaze , and examination can reveal a slow ipsilateral lateral rectus movement [ 4 , 5 ] . ms has been implicated as the cause of unilateral abducens palsy in 49% of cases , though mri may not detect brain stem lesions in all cases . one 2002 study investigating nontraumatic causes of sixth nerve palsies in patients 2050 years of age found ms to be the cause in 24% of cases . in addition to ms , the differential diagnosis for an abducens nerve palsy includes mass lesions , lyme disease , viral infection , syphilis , sarcoidosis , and vascular disease . here , we report a patient who presented with a unilateral isolated abducens palsy as the initial sign of ms . a 28-year - old man with a past medical history of hypertension and obesity presented to the emergency department with a 1-day history of double vision , most prominent upon left lateral gaze . the double vision resolved with covering either eye . in addition , he had a 12-week history of paresthesia of the distal right hand and forearm , and the fifth digit of the right foot . initial laboratory findings revealed only a mild leukocytosis ( 11.3 10/l ) and no other hematologic or electrolyte abnormalities . several focal areas of increased t2 signal intensity were noted within the periventricular white matter of the frontal and parietal lobes , subcortical white matter of the left temporal lobe , and the left pons . these findings likely represented multiple lesions distributed in both time and space , and , along with the patient 's clinical history and examination , were suggestive of a diagnosis of ms . the patient was admitted and started on intravenous methylprednisolone . further workup to rule out other etiological causes of illness revealed an elevated esr of 25 mm / h , negative viral and lyme serologies , nmo igg negative and a negative ana . he was treated with methylprednisolone 500 mg intravenously for 3 days and then discharged home . he was re - evaluated in the office 2 days after hospital discharge and had complete resolution of the cn vi palsy . isolated nerve palsies occur in only 10.4% of patients with ms , with abducens palsy as the third most common isolated nerve palsy , occurring in 1.0% of patients with ms , behind trigeminal ( 4.8% ) and facial ( 3.7% ) . these palsies occur most commonly at disease onset , though they can also occur during the course of the disease . the lesion in the medial pons was enhancing and thus consistent with an active lesion , and the location was consistent with his cn vi palsy . the sixth nerve nucleus is located in the pontine tegmentum , and a compact fiber tract bundle containing motor neurons from this nucleus runs medially towards the ventral region , where the cranial nerve exits the pons [ 3 , 8 ] . our patient 's pontine lesion appeared to be located along the fiber tract emerging from the sixth nerve nucleus , and correlates with his diplopia . while an mri lesion was detected clearly in this case , it should be noted that previous authors have found that mri does not always detect brain stem lesions accounting for these palsies in ms patients [ 4 , 6 ] . in any patient presenting with isolated cranial nerve palsies , ms must be considered within the differential diagnosis . in patients younger than 50 years of age ( as with our patient ) , infectious causes ( lyme disease , viral infections , syphilis ) , sarcoidosis , and autoimmune vasculitis should also be considered on the differential diagnosis , whereas for older patients small vessel vascular disease should be considered as well [ 7 , 8 ] . the presence of multiple presenting neurological deficits including cranial nerve palsies is suspicious for ms and should be investigated with mri . though mri may not always detect brain stem lesions responsible for cranial nerve palsies , it can reveal other white matter lesions of the cns that can aid in the diagnosis of ms , as was the case with our patient .

for a prospective study of lead exposure and early development , we recruited pregnant women from a lead smelter town and from an unexposed town in yugoslavia and followed their children through 7 years of age . in this paper we consider associations between lifetime lead exposure , estimated by the area under the blood lead ( bpb ) versus time curve ( auc7 ) , and intelligence , with particular concern for identifying lead 's behavioral signature . the wechsler intelligence scales for children - version iii ( wisc - iii ) was administered to 309 7-year - old children , 261 of whom had complete data on intelligence , blood lead , and relevant sociodemographic covariates ( i.e. , home observation for the measurement of the environment ( home ) , birth weight , gender , sibship size , and maternal age , ethnicity , intelligence , and education ) . these showed anticipated associations with 7-year intelligence , explaining 41 - 4% of the variance in full scale , performance , and verbal iq . before covariate adjustment , auc7 was unrelated to intelligence ; after adjustment , auc7 explained a significant 2.8%-4.2% of the variance in iq . after adjustment , a change in lifetime bpb from 10 to 30 micro / dl related to an estimated decrease of 4.3 full scale iq points ; estimated decreases for verbal and performance iq were 3.4 and 4.5 points , respectively . auc7 was significantly and negatively related to three wisc - iii factor scores : freedom from distractibility , perceptual organization , and verbal comprehension ; the association with perceptual organization was the strongest . consistent with previous studies , the iq / lead association is small relative to more powerful social factors . findings offer support for lead 's behavioral signature ; perceptual - motor skills are significantly more sensitive to lead exposure than are the language - related aspects of intelligence.imagesfigure 1 . afigure 1 . bfigure 1 . cfigure 1 . dfigure 2 . while brain stem involvement in multiple sclerosis ( ms ) is relatively common , isolated cranial nerve palsies are rare , especially when they represent the initial presenting sign of a new diagnosis of ms . this report describes a patient with no prior history of ms whose sole presenting sign was an isolated abducens palsy . an enhancing pontine lesion was found on mri which correlated with his abducens palsy , and additional nonactive lesions on mri led to a diagnosis of ms . this case demonstrates the importance of considering ms as part of the differential diagnosis of patients with isolated cranial nerve palsies .

according to the world health organization ( who ) , a maternal death is defined as death of a woman while pregnant or within 42 days of termination of pregnancy , irrespective of the duration and site of pregnancy , from any cause related to or aggravated by pregnancy or its management ( icd-10 ) . about 99% of these women are from developing world with over 90% concentrated in africa and asia . the risk of a woman dying as a result of pregnancy and childbirth during her lifetime is about 1 in 6 in afghanistan compared with 1 in 30,000 in northern europe . united nation ( un ) report card on millennium development goal-5 concluded that little progress had been made in sub - saharan africa where half of all maternal deaths take place . the progress shown by the south asian countries including india which accounts for 25% of all maternal deaths is also not impressive . most of the evidence for maternal mortality is obtained through hospital data and community based reports , which are situated mostly in urban areas , whereas most of the maternal deaths are from rural areas . this study was done to assess maternal mortality in a tertiary medical college hospital situated in semi urban part of western maharashtra where large numbers of patients are referred from rural parts of western maharashtra and north karnataka . this study was done to assess the local causes of maternal mortality and suggest remedial measures to reduce the same . to calculate the maternal mortality rate in our hospital.to assess the epidemiological aspects of maternal mortality.to assess the causes of maternal mortality.to suggest ways to reduce the mmr . to calculate the maternal mortality rate in our hospital . to assess the epidemiological aspects of maternal mortality . to assess the causes of maternal mortality . to suggest ways to reduce the mmr . to calculate the maternal mortality rate in our hospital.to assess the epidemiological aspects of maternal mortality.to assess the causes of maternal mortality.to suggest ways to reduce the mmr . to calculate the maternal mortality rate in our hospital . to assess the epidemiological aspects of maternal mortality . to assess the causes of maternal mortality . to suggest ways to reduce the mmr our hospital is a semi - urban , tertiary care center ; situated in the southernmost part of western maharashtra . it gets a large number of referrals from maternity homes , primary health centers from rural parts of western maharashtra , and also from north karnataka . the present study was a retrospective study , conducted in the department of obstetrics and gynecology of this hospital . data regarding maternal mortality was collected from maternal mortality register after obtaining permission from the medical superintendent of the hospital . the details of maternal deaths from january 2001 to december 2010 were collected and analyzed with respect to following epidemiological parameters . locality wise distribution of maternal deaths.literacy wise distribution of maternal deaths.gravidity wise distribution of maternal deaths.maternal deaths according to receipt of antenatal care.distribution of maternal deaths according to socioeconomic status.causes of maternal deaths . the details of number of live births from january 2001 to december 2010 were collected from labor ward register . maternal mortality rate for the study period was calculated by using the formula- mean maternal mortality ratio for the study period was calculated by calculating the mean of yearly mmr of the entire study period . during the study period , january 2001 to december 2010 , there were a total of 39704 live births and 120 maternal deaths . maximum maternal deaths ( 49.16% ) were reported in the age group of 20 to 24 years . ( 43.33% ) , more maternal deaths were reported in women from rural areas ( 69.16% ) as compared to women from urban areas ( 30.83%).maximum maternal deaths were reported in unbooked patients ( 83.33% ) as compared to booked patients ( 16.66% ) . epidemiological characteristics of maternal deaths ( n = 120 ) in the study period , 72.5% of maternal deaths were due to direct causes . year - wise distribution of direct causes of maternal deaths is shown in table 2 . the classical triad of hemorrhage ( 26.66% ) , eclampsia ( 26.66% ) , and sepsis ( 18.33% ) was the major direct causes of maternal deaths , whereas only one maternal death ( 0.83% ) was due to obstructed labor . in the study period , anemia , jaundice , and heart disease accounted for 10% , 9.16% , and 3.33% of maternal deaths respectively and miscellaneous cause like acute gastroenteritis accounted for 5% of maternal deaths as shown in table 3 . high incidence of maternal deaths reflects poor quality of maternal services , late referral and low socioeconomic status of the community . various studies done in india in the last 15 years have shown wide variation in mmr ranging from 47/100000 to 625/100000 births.[49 ] madhu jain has reported a very high mmr of 2270/100000 . this study has comparatively high mmr , which could be due to the fact , that our hospital is a tertiary care hospital and receives a lot of complicated referrals from rural areas of southern maharashtra and also from north karnataka at a very late stage . in our study , 70% of maternal deaths were in the age group of 20 to 29 years , as highest numbers of births are reported in this age group . more maternal deaths were reported in women from rural areas ( 69.16% ) , unbooked patients ( 83.33% ) , illiterate women ( 65% ) , and women belonging to low socioeconomic status . ( 83.33% ) all our findings were similar to studies by jain , jadhav , pal , onakewhor . in our study , hemorrhage ( 26.66% ) , eclampsia ( 26.66% ) , and sepsis ( 18.33% ) were the major direct causes of maternal deaths . our findings were consistent with studies by jain , jadhav , pal , onakewhor , and shah . even today large number of maternal deaths is due to the classical triad of hemorrhage , sepsis , and eclampsia . all these are preventable causes of maternal mortality provided the treatment is instituted in time . unfortunately , in many cases , patients were referred very late , in critical condition , unaccompanied by health care worker . many patients had to travel a distance of 70 to 80 kilometers in a private vehicle to reach our tertiary center . most of these deaths are preventable if patients are given appropriate treatment at periphery and timely referred to higher centers . training of medical officers and staff nurses working in rural areas by programs like basic emergency obstetrics care ( bemoc ) and skilled attendant at birth ( sab ) training gives a ray of hope of reducing maternal mortality . anemia , jaundice , and heart disease were responsible for 10% , 9.16% , and 3.33% of maternal deaths , respectively . these findings were consistent with studies by jain , jadhav , pal , and onakewhor . maternal deaths can be prevented by improving the health care facilities in rural areas by ensuring round the clock availability of certain basic drugs like injection magnesium sulfate , tablet misoprostol as most maternal deaths in rural areas are still due to eclampsia and post partum hemorrhage . early detection of high risk pregnancies and referring them to a tertiary center at the earliest can reduce the complications of high risk pregnancies . national rural health mission ( nrhm ) can play a major role in reducing maternal mortality by advocating institutional deliveries and timely referral of high risk cases . although we have not actually evaluated the impact of aforementioned educational programs on maternal mortality , it would be interesting to direct future studies in this regard . even today most maternal deaths are seen in patients from rural areas , unbooked , illiterate patients and patients from low socioeconomic status . hemorrhage , eclampsia and sepsis are the major causes of maternal deaths . improvement in primary health care in rural areas and proper implementation of nrhm programs and up gradation of hospitals in rural areas can definitely bring down the number of maternal deaths . head and neck squamous cell carcinoma ( hnscc ) is the sixth most common cancer worldwide , with an average 5-year survival rate of around 50% ( 1 ) . despite improvements in the therapeutic modalities , the prognosis of these patients has not been significantly improved in the last three decades . alcohol and both smoked and smokeless tobacco use are associated with increased risk of developing malignancy of the oral cavity and pharynx ( 2 ) . recently , the role of oncogenic viruses especially hpv in the pathogenesis of scc has attracted the researchers attention . hpv viral proteins , e6 and e7 , play major roles in the cervical as well as orophararyngeal carcinogenesis . this proteins act through inactivation of tumor suppressor genes , p53 and retinoblastoma ( rb ) genes , resulting in cell immortalization and transformation ( 4 ) . perhaps one of the most important developments in the head and neck oncology over the past decade is the recognition of hpv status in oropharyngeal sccs ( opsccs ) . it is obvious that hpv - ralated opscc has unique epidemiological and clinical entity and it had better distinguishes from the ocsccs because of unknown role of hpv in non - oropharyngeal head and neck cancers ( 5 - 7 ) . ( 9 ) , and lingen et al . in 2013 ( 10 ) considered low etiologic role for hpv status in ocsccs . mentioned that probably other risk factors that are still unknown play roles in the pathogenesis of ocsccs . in recent years , much attention has been paid to the prognostic role of hpv status in hnscc . some researchers have found that patients with hpv detected in their cancers , have better prognosis compared to those without hpv ( 11 - 15 ) . nevertheless there are studies that have found no difference or poorer prognosis for hpv related tumors ( 16 - 22 ) . in this study , we have investigated the prevalence rate of hpv status in patients with ocscc , and its significance in the clinicopathological characteristics of tumors and patients prognosis . 132 patients with diagnosis of ocscc were included in this study between 2001 and 2013 . their records were collected from the files of department of pathology , faculty of dentistry and omid hospital , mashhad university of medical sciences , iran . age , gender , anatomical region , tnm stage , histological grade , therapeutic interventions , events including local , distant relapse and death were extracted from the medical records database . the haematoxylin and eosin stained sections from all specimens were reviewed by two pathologists to confirm the histology and grading . histological grading was assessed according to the who guidelines and tumor staging was reported according to the american joint committee on cancer staging criteria ( ajcc sixth edition , 2002 ) . cleaning off the microtome blade with xylene and ethanol after cutting each paraffin block was taken for preventing and monitoring contamination of samples . the tumor samples were deparaffinized using a series of xylene and ethanol washes . in this study , the tissue sample was mixed with 400 l digestion buffer which consisted of tris - cl ( 100 mm , ph 7.5 ) and tween-20 ( 0.055 ) and proteinase k 3l ( 20 mg / ml ; frementase , germany ) , followed by incubation at 56c for 3 hour . after the complete digestion of tissue , the mixture was incubated at 100c in hotblock ( accublock digital dry baths ; usa ) to inactivate proteinase k. the samples were then centrifuged at 5000 rpm , and the supernatant containing dna was used for pcr amplification ( 96-well thermal cycler ; applied biosystem , usa ) . the quality of dna was determined by amplification of housekeeping gene ( -globin ) using the gh20/pc04 primers ( cinnagen , iran ) . a consensus sequence from the l1 region of the hpv genome was amplified using gp5+/gp6 + primers ( cinnagen , iran ) ( 2 ) . gp5:5-ttt gtt act gtg gta gat act ac-3 and gp6:5-gaa aaa taa act gta aat cat att c-3 were used as the primer set . hpv typing ( hpv 6 , 11 , 16 , 18 and 31 ) was also performed using hpv type specific primers ( cinnagen , iran ) ( 19 ) . pcr products were separated in 2% agarose gels and visualized by green veiwer staining ( parstous , iran ) . all analyses were performed using data processing program spss / pc version 16.0 for windows ( spss inc . , chicago , il , usa ) . chi square , mann -whitney , student s t test and fisher s exact tests were used to assess the statistical significant differences between hpv status and clinicopathological parameters . the os and dfs rates were estimated by the kaplan - meier method , and differences in survival rates were analyzed with the log - rank test . 5 to 10 m sections were cut from each paraffin block . cleaning off the microtome blade with xylene and ethanol after cutting each paraffin block the tumor samples were deparaffinized using a series of xylene and ethanol washes . in this study , the tissue sample was mixed with 400 l digestion buffer which consisted of tris - cl ( 100 mm , ph 7.5 ) and tween-20 ( 0.055 ) and proteinase k 3l ( 20 mg / ml ; frementase , germany ) , followed by incubation at 56c for 3 hour . after the complete digestion of tissue , the mixture was incubated at 100c in hotblock ( accublock digital dry baths ; usa ) to inactivate proteinase k. the samples were then centrifuged at 5000 rpm , and the supernatant containing dna was used for pcr amplification ( 96-well thermal cycler ; applied biosystem , usa ) . the quality of dna was determined by amplification of housekeeping gene ( -globin ) using the gh20/pc04 primers ( cinnagen , iran ) . a consensus sequence from the l1 region of the hpv genome was amplified using gp5+/gp6 + primers ( cinnagen , iran ) ( 2 ) . gp5:5-ttt gtt act gtg gta gat act ac-3 and gp6:5-gaa aaa taa act gta aat cat att c-3 were used as the primer set . hpv typing ( hpv 6 , 11 , 16 , 18 and 31 ) was also performed using hpv type specific primers ( cinnagen , iran ) ( 19 ) . pcr products were separated in 2% agarose gels and visualized by green veiwer staining ( parstous , iran ) . all analyses were performed using data processing program spss / pc version 16.0 for windows ( spss inc . , chicago , il , usa ) . chi square , mann -whitney , student s t test and fisher s exact tests were used to assess the statistical significant differences between hpv status and clinicopathological parameters . the os and dfs rates were estimated by the kaplan - meier method , and differences in survival rates were analyzed with the log - rank test . moreover , eight patients were excluded because their clinicopathological databases were not complete . remaining 114 ocscc cases comprised of 58 men and 56 women . the mean age of patients was 58.6 years ( range 19 - 85 years).the follow - up period ranged from 6 months to 8 years . the hpv sequences were detected in 15 patients ( 13.16% ) : 5 cases with hpv11 ( 33.3% ) , one case with hpv6 ( 6.7% ) and two cases with both hpv6 and 11 ( 13.3% ) . in 7 patients ( 46.7% ) patients characteristics and tumor features of the hpv positive and negative ocscc cases are compared in table 1 . no significant differences were observed between the two groups in the variables of age , gender , tumor site , size and histological grading . fifty five hpv - negative patients ( 55.6% ) were n0 , while five hpv - positive patients ( 33.3% ) were n0 . in addition , while twenty two hpv - negatives ( 22.2% ) were n2 , eight hpv - positive patients ( 53.3% ) were n2 . the median lymph node involvement was significantly higher in hpv positive group compared to hpv negative group ( p = 0.045 ) . as a result , significantly more stage iva was seen among the hpv - positive tumors compared to the hpv - negative ones ( 66.7% vs. 35.4% , p = 0.04 ) . most of the hpv positive and negative cases were treated with surgery and adjuvant radiation therapy ( table 2 ) . thirty four out of 99 hpv - negative patients ( 34.3% ) died from their disease compared with 11 out of 15 in the hpv - positive cases ( 73.3% ) . chi square test revealed a significant relation between the rate of death and hpv positive status ( p = 0.004 ) . survival and hpv dna status : the median dfs was 13.9 months among the hpv - positive and 49.9 months among the hpv - negative patients with a significant difference ( p = 0.029 ) . also the median os of 15.7 months in the hpv - positive group compared to 49.6 months in the hpv - negative group was significantly better ( p = 0.017 ) . the kaplan - meier survival curves for the hpv - positive and negative cases are shown in figure 1 . we used multivariate analysis involving cox proportional hazards model to determine which factors were jointly predictive of os and dfs . the hpv status ( hr = 2.10 , ci = 1.02 to 4.28 , p = 0.041 ) and stage ( hr = 1.92 , ci = 1.36 to 2.70 , p < in addition , both hpv status ( hr = 2.80 , ci = 1.35 to 5.79 , p = 0.005 ) and stage of disease ( hr = 1.95 , ci = 1.37 to 2.79 , p < moreover , eight patients were excluded because their clinicopathological databases were not complete . remaining 114 ocscc cases comprised of 58 men and 56 women . the mean age of patients was 58.6 years ( range 19 - 85 years).the follow - up period ranged from 6 months to 8 years . the hpv sequences were detected in 15 patients ( 13.16% ) : 5 cases with hpv11 ( 33.3% ) , one case with hpv6 ( 6.7% ) and two cases with both hpv6 and 11 ( 13.3% ) . in 7 patients ( 46.7% ) patients characteristics and tumor features of the hpv positive and negative ocscc cases are compared in table 1 . no significant differences were observed between the two groups in the variables of age , gender , tumor site , size and histological grading . fifty five hpv - negative patients ( 55.6% ) were n0 , while five hpv - positive patients ( 33.3% ) were n0 . in addition , while twenty two hpv - negatives ( 22.2% ) were n2 , eight hpv - positive patients ( 53.3% ) were n2 . the median lymph node involvement was significantly higher in hpv positive group compared to hpv negative group ( p = 0.045 ) . as a result , significantly more stage iva was seen among the hpv - positive tumors compared to the hpv - negative ones ( 66.7% vs. 35.4% , p = 0.04 ) . most of the hpv positive and negative cases were treated with surgery and adjuvant radiation therapy ( table 2 ) . thirty four out of 99 hpv - negative patients ( 34.3% ) died from their disease compared with 11 out of 15 in the hpv - positive cases ( 73.3% ) . chi square test revealed a significant relation between the rate of death and hpv positive status ( p = 0.004 ) . survival and hpv dna status : the median dfs was 13.9 months among the hpv - positive and 49.9 months among the hpv - negative patients with a significant difference ( p = 0.029 ) . also the median os of 15.7 months in the hpv - positive group compared to 49.6 months in the hpv - negative group was significantly better ( p = 0.017 ) . the kaplan - meier survival curves for the hpv - positive and negative cases are shown in figure 1 . we used multivariate analysis involving cox proportional hazards model to determine which factors were jointly predictive of os and dfs . the hpv status ( hr = 2.10 , ci = 1.02 to 4.28 , p = 0.041 ) and stage ( hr = 1.92 , ci = 1.36 to 2.70 , p < in addition , both hpv status ( hr = 2.80 , ci = 1.35 to 5.79 , p = 0.005 ) and stage of disease ( hr = 1.95 , ci = 1.37 to 2.79 , p < the participation of hpv in the oral and oropharyngeal carcinogenesis was first proposed in 1983 by syrjanen et al . ( 23 ) and then supported by several other studies . according to many studies , the hpv prevalence rate in oral cancer is varied between 0% - 100% ( 6 , 19 , 24 - 26 ) . this widespread variability can be in part due to differences in methodology of hpv detection ways and differences in sample types , populations , and anatomic sites tested ( 27 , 28 ) . moreover , various studies have reported conflicting results for the probable role of hpv on the patient 's prognosis ( 15 , 16 , 29 ) . in the current study , regarding the sample size , the majority of small to medium sized studies ( < 100 patients ) have revealed a wide variation in hpv prevalence rates ( varying between 0% and 100% ) ; however , larger studies generally tend to show lower hpv prevalence rates ( varying between 1.4% and 48.8% ) . ( 27 ) have mentioned the sample size as an important factor in the reportedly heterogeneous prevalence rate of hpv , which is in accordance with our findings . the hpv positivity rate of 13.6% in our study is almost similar to those other reports of oral cavity cancers which are ranged from 12% to 70% for opscc ( 2 , 15 , 22 , 30 ) versus 3% to 15% for ocscc ( 2 , 20 , 31 ) . while there is another report that mentions a 25% incidence rate of hpv among tongue cancers in iran and yet all of them subtypes of 18 and 16 ( 32 ) , in the current study , 15 cases ( 13.16% ) were identified as hpv positive and the detected viral subtypes were subtypes 6 and 11 . however , it is not still clear whether these subtypes result in an infection driven carcinogenesis or they are just a transient infection ( 5 , 33 ) . pannone et al . mentioned that hpv 6 , which was known as low risk or non - oncogenic subtype to the cervix , was present in a larger number of head and neck cancers . hpv 6 and 11 have been found in some tonsillar and laryngeal carcinomas and in the malignant transformation of benign laryngeal papillomas , hpv 11 has been most commonly implicated . hpv 6 and 11 have also been on suspicion of malignancies such as verrucous carcinoma of the oral cavity ( 34 , 35 ) . hpv 6 and 11 were also observed in other studies on cancers of the head and neck ( 36 , 37 ) . studies on opscc often suggested that the incidence of hpv in this region is more prevalent ( 5 , 33 , 38 ) . it is also mentioned that hpv - mediated opsccs are more likely late stage , poorly differentiated , and with large lymph nodes in the neck , and in spite of that they have less risk of recurrence and death ( 2 , 5 , 24 , 33 , 39 - 41 ) . some studies indicate that such hpv - positive tumors are more radio - sensitive and have better overall survival rate ( 33 , 42 , 43 ) . these patients tend to be younger and often male with less consumption of alcohol and smoking ( 33 , 41 , 44 ) . there is also data indicating that patients with hpv - positive opscc are associated with a higher number of sexual partners ( 33 , 34 ) . in sccs limited to the oral cavity , usually the hpv prevalence rate is lower and hpv16 is also the most common type detected ( 2 , 28 ) . some studies have suggested that hpv does not play a significant role in the etiology of ocscc ( 8 - 10 ) and also does not play a role in the progression toward malignancy , even though some studies have demonstrated the role of hpv16 and 18 in oral carcinogenesis ( 45 - 47 ) . notably , because of relatively low frequency of hpv in ocsccs , only few studies have attamped to correlate hpv status with clinical outcome ( 5 , 24 , 46 , 47 ) . in most of the studies of hnsccs , the demonstrated clinicopatological features about hpv positive cases are usually related to the oropharyngeal samples ; therefore , little is known about the influence of hpv on clinical course of patients with the oral cavity cancer because of the lower hpv prevalence . no data currently supports the idea that hpv is significantly associated with improved outcome for patients with oral cancer . only few published studies on patients with oral cavity carcinoma specifically examined the impact of hpv on outcome . kaminagakura studied 114 patients and found a nonsignficant trend towards improved survival for 22 hpv - positive patients ( 47 ) . sugiyama et al . demonstrated a nonsignficant trend towards improved overall survival for hpvpositive oral cavity cancer patients ( 48 ) . smith found no association with hpv and outcome for patients with oral carcinoma , based on either serology or tumor hpv detection ( 49 ) . also in isayeva s cohort study of 89 patients with oral cavity carcinoma , no significant association was found for patients with either hpv16/18 e6e7 rna and time to disease progression or disease specific survival ( 5).therefore , future studies on oral cavity scc should be powered to address the important clinical issue of hpv status . in the present study , the mean age of the cases was 58.6 years , and there was no statistical difference between the hpv - positive and the hpv - negative groups based on age . however , in some studies , the hpv - associated patients who suffer from oropharyngeal cancers tend to be younger ( 33 , 39 , 42 , 44 ) . in our study , 53.3% of hpv - positive patients were males and 46.7% were females , and there was no meaningful relation between gender and the hpv status . such findings have been observed in some other studies ( 29 ) , although some studies on oropharynx lesions have reported that the hpv - positive cases are predominantly male ( 33 , 41 , 42 , 44 ) . in the present study , the most common tumor site was tongue , and there was no meaningful statistical relation between tumor location and the hpv status . similar findings have been reported in other studies ( 19 , 29 ) . according to the tumor grade , although 60 % of the hpv - positive patients were grade 2 and 59.6% of hpv - negative patients were grade1 , no meaningful statistical difference was found between hpv status and histological grading of the tumor ( 16 , 19 ) . although in most of the oropharyngeal cancer studies advanced tumor stages have been observed in the hpv - positive cases ( 18 , 33 , 39 , 41 , 44 , 50 ) , there are some studies which have not seen any difference ( 16 , 19 , 32 ) . in this study 55.6 % of the hpv - negative patients and only 33.3% of the hpv - positive patients were n0 . moreover , 66.7% of the hpv positive groups and only 35.4% of the hpv negative ones were stage iva . ( 51 ) have reported similar findings , although in some studies no difference have been observed ( 13 ) and in most of the hpv - related opscc cases higher lymph node involvement have been observed ( 18 , 33 , 39 ) . in the current study local recurrence was the most commen type of relapse , and there was no statistically significant difference according to hpv status . in most of the hpv - related opscc studies low reccurence rate have been observed ( 2 , 50 ) , although weinberger et al . have noted that hpv status has no prognostic value for local recurrence alone ( 52 ) . in our study , 34.4% of the hpv - negative patients and 73.3% of the hpv - positive patients died of disease . that statistically meaningful relation was observed between hpv status and patients death . less mortality risk in hpv - positive cases but as far as we know , no study on oral cavity cancers has especially dealt with the mortality rate of hpv - positive patients . we found that hpv - negative patients survived significantly longer than hpv - positive patients when both the os and the dfs were measured . similar findings have been reported in other studies based on tumors with higher stages of disease ( 16 , 22 ) . while the majority of oropharyngeal studies have reported an improved prognosis of hpv positive tumors ( 2 , 5 , 18 , 19 , 33 , 39 - 41 , 44 , 50 , 53 ) , there are still other studies which have reported no difference ( 2 , 5 , 18 , 19 ) . in our study there are other studies which indicate hpv - related opscc patients , have a lower intake of tobacco and alcohol ( 33 , 39 , 44 , 50 ) . currently , little is known about the influence of hpv on the clinical course and survival of patients with ocscc . although the findings of this study revealed relatively low prevalance of hpv dna in ocsccs and also the well known high risk subtypes were not observed , the carcinogenic role of this virus and its less common variant of hpv 6 and 11 , which we did see in the current study , can not be totally ignored in the oral cavity cancers . regarding high mortality rate of these variants of hpv - positive tumors , further investigations with higher sample volume using more developed methods currently , little is known about the influence of hpv on the clinical course and survival of patients with ocscc . although the findings of this study revealed relatively low prevalance of hpv dna in ocsccs and also the well known high risk subtypes were not observed , the carcinogenic role of this virus and its less common variant of hpv 6 and 11 , which we did see in the current study , can not be totally ignored in the oral cavity cancers . regarding high mortality rate of these variants of hpv - positive tumors , further investigations with higher sample volume using more developed methods are still required .

background : epidemiological data pertaining to maternal mortality is valuable in each set up to design interventional programs to favourably reduce the ratio . this study was done to evaluate the maternal mortality rate in our hospital , to assess the epidemiological aspects and causes of maternal mortality , and to suggest recommendations for improvement.methods:this was a 10 year retrospective study . epidemiological data was collected from the hospital register and maternal mortality ratio , epidemiological factors and causes affecting maternal mortality were assessed.results:a total of 120 maternal deaths occurred . most maternal deaths occurred in the age group of 2024 years , multiparous women ( 56.66% ) , women from rural areas ( 69.16% ) , illiterate women ( 65% ) , unbooked patients ( 83.33% ) , and patients of low socioeconomic status ( 83.33% ) . direct causes accounted for 72.5% of maternal deaths where as 27.5% of maternal deaths were due to indirect causes.conclusion:there is a wide scope for improvement as a large proportion of the observed deaths are preventable . backgroundsquamous cell carcinoma ( scc ) is the most common malignancy of the oral cavity . a relationship between the human papilloma virus ( hpv ) infection and the prognosis of oral cavity scc ( ocscc ) has been discussed before.objectiveswe investigated the prevalence rate of hpv status in patients with ocscc , and its effects on clinicopathological characteristics of tumors and patients prognosis.patients and methodssections of formalin - fixed , paraffin - embedded tissue blocks from 114 histopathologically confirmed ocscc cases were investigated in this study . polymerase chain reaction ( pcr ) was applied to evaluate the hpv status in the samples.resultsfifteen ( 13.16% ) cases were identified as hpv positive . the detected viral subtypes in this study were the subtypes 6 and 11 . the stage and especially lymph node stage was significantly higher in the hpv positive group compared to the hpv negative group ( p = 0.04 ) . disease free survival ( dfs ) was remarkably lower in the hpv positive group compared to the hpv negative group ( 13.9 vs. 49.9 months , p = 0.02 ) . overall survival ( os ) was also significantly inferior in the hpv positive group ( 15.7 vs. 49.6 months , p = 0.01 ) . in the current study , no significant differences were observed between two groups in relation to the variables of age , gender , tumors site , tumor size , tumor grading and also the recurrence rate.conclusionsthe observed higher mortality rate among the hpv positive group indicates the poorer prognosis of this group in comparison with the hpv negative patients . the incidence rate of hpv infection was low in the studied samples ; however , interaction of subtypes 6 and 11 of hpv in poorer prognosis of the patients and a carcinogenic role of hpv in ocscc can not be ruled out .

it is estimated that depression will become the second most common cause of disability , next to heart disease in a few decades . major depression affects 1 in 20 people during their lifetime . in many cultures , especially in developing eastern countries , talking about emotions somatic metaphors and complaints are usually expressed as substitutes for emotional discharge . such variability in depression rates , as noted by simon and his colleagues , may represent problems with definition and measurement rather than true differences in prevalence . it seems that western measures are not reliable sources for the estimation of the prevalence of affective disorders in culturally divergent populations . as kleinman noted , depression is not a universal psychiatric construct and the ways of understanding the body and the self are so different that this may lead to differences in psychopathology . neurasthenia refers to a bodily state and for the chinese it is easier to talk about somatic complaints rather than one s emotional status . one reason for this apparent disregard may be the stigma that is attached to psychiatric symptoms in chinese culture , compared to the relative acceptance of physical complaints . the who collaborative study assessed 573 patients , and showed that feelings of guilt and self - reproach were commonest in basle and montreal and least in tehran , where suicidal ideation was rare . in contrast , somatic symptoms were commonest in tehran and least in basle and montreal . in depressed chinese american patients , researchers found that the most common presenting complains were fatigue , insomnia , headache , cough and pain . in chinese culture in which psychiatric symptoms are usually stigmatized , somatic symptoms are accepted more than direct presentation of emotional symptoms . patients exhibited less psychiatric symptoms , when referred to a private physician , than those who were visited by a general primary care physician . luis caballero and his colleagues studied a population of spanish patients with major depressive disorders . they observed that 93% of patients had at least one somatic symptom which was fully or partially attributed to depression . bhui revealed in his study that south asians were more likely to visit their general practitioners ( gps ) , and exhibited somatic manifestations of mental distress more commonly than other groups . they were less likely to have a recognized mental disorder than white groups , and even with recognizable mental disorder , they were least likely to be referred to specialist care by gps . on the other hand , some surveys showed no differences in psychological and somatic symptoms between western and other countries . in one study two thirds of depressed patients in primary care presented with somatic symptoms . in another study , the complaints expressed by the patients as somatic entities , were actually depression symptoms in dsm - iv . in a study of 504 patients in tehran , researchers found a high rate of somatic symptoms in depressed patients . also , a similar study in kerman on 246 depressed patients , with frequent symptoms , showed that 40% of case suffered from somatic symptoms . according to the above - mentioned studies and our clinical experience , it seems that depression as a disorder has a different picture in eastern cultures compared to the one delineated by western researchers . in regard to the more frequent expression of somatic symptoms in eastern cultures , we assessed the probability of different presentations of depressive signs and symptoms in male and female patients and in cases with different cultural backgrounds and education . such studies can lead to a better understanding of such disorders and more accurate diagnosis of depression in developing countries . the definition and differentiation of psychiatric problems presented as somatic symptoms may prevent unnecessary interventions and expenses . mental health professionals working in such cultural atmosphere have frequently witnessed improvement in patients on antidepressant medications . these patients often had histories of traveling far and wide in search for a solution to their suffering which eventually has ended in the psychiatrist office . in this study , we have tried to answer some questions about the relationship between cultural background , level of education , age , marital status and gender on the one hand , and presenting symptoms of depressed patients on the other . this cross - sectional descriptive study assessed the presenting symptoms of 500 patients with major depressive disorder referred to a psychiatric clinic affiliated with shiraz university of medical sciences . diagnosis of the disorder was made based on the dsm - iv - tr ( diagnostic and statistical manual of mental disorders - iv - text revision ) criteria according to a face to face structured psychiatric interview based on the american psychiatric association criteria for the diagnosis of major depressive disorder . this is a standard tool for the diagnosis of such disorder in many countries including iran and conducted by a bilingual psychiatrist with more than 15 years of experience . the criteria for recruiting the patients in the study included no history of current substance abuse or dependency , patient s symptoms could not be justified by a general medical condition or other psychiatric entities such as anxiety or somatoform disorders , receiving no active psychiatric treatment , not visited by a psychiatrist during 3 months before evaluation , and not taking any psychiatric medications during this time . dsm - iv classifies the signs and symptoms of depression into two groups of vegetative and mental conditions . since pain is assessed as a frequent vegetative symptom , it was considered as a separate entity and eventually categorized into three groups including mental symptoms , pain , and physical symptoms other than pain . through psychiatric interview , we assessed the presenting complaints of these patients and considered such symptoms as guilt feeling , hypochondriac status , insomnia , and losses of appetite , weight , concentration , and interest , suicidal idea and attempt , hopelessness and crying . also the patients were asked about their chief complaints which were categorized into mental symptoms with pain , and physical symptoms without pain , the duration of the illness , description of the emotional or somatic disorders by the patients , causes of illness expressed by the patients as family problems including interpersonal or intergenerational difficulties , living problems such as occupational or financial difficulties , losses incurred , marital conflicts , and romantic breakups , and the clinician who first visited the patient namely general practitioner , psychiatrist or other specialist . these variables were compared by proper statistical analysis ( chi - square ) according to sex , age , marital status , cultural background ( rural or urban ) and education level . logistic regression analysis , backward method , was used to compare the association of different independent variables in patients with or without somatization as a symptom . of 500 patients studied , 380 ( 76.0% ) were females , 356 ( 71.2% ) married , 246 ( 46.2% ) had elementary school education , and 306 ( 61.2% ) belonged to the urban cultural background . demographic characteristics of the patients tables 2 and 3 demonstrate the descriptive features of patients chief complaints and symptoms of which headache ( 15.2% ) was the most frequent complaint , followed by irritability ( 10.6% ) , pain in the other areas of the body ( 10.4% ) , depression ( 8.0% ) and forgetfulness ( 7.2% ) . among physical ( organic ) complaints , the gastrointestinal ( 7.0% ) symptoms were more frequent than respiratory ( 3.4% ) and cardiac symptoms ( 3.2%).in this context , pain and physical complaints were more common in persons with lower education ( p<0.001 ) , rural cultural background ( p<0.001 ) , women ( p<0.001 ) and married patients ( p=0.007 ) . individuals with higher education ( p<0.001 ) and urban cultural background ( p<0.001 ) were more likely to visit a psychiatrist . there was no significant difference in the referral to psychiatrist between age , sex and marital status . frequency of chief complaints description of clinical variables hypochondriac obsessions were more common in the married ( p=0.024 ) , elder ( p<0.001 ) and lower educated people ( p<0.001).insomnia was more common in people with rural cultural background ( p=0.009 ) and in individuals with lower education ( p<0.001 ) . guilt feeling was more common in the older and younger people compared to middle aged patients ( p<0.001 ) . loss of appetite was more prevalent among older and younger people compared to middle aged patients ( p=0.017 ) and in people with rural cultural background ( p=0.003 ) . suicidal ideation were present more frequently among married persons ( p<0.001 ) , the lower age group ( p<0.001 ) and people with rural cultural background ( p=0.027 ) . suicidal attempt was more frequent in the lower age group ( p=0.009 ) and in the married ones ( p=0.047 ) . our study showed no difference in suicidal ideation and suicidal attempt between men and women . people with lower education ( p<0.001 ) and with rural cultural background ( p=0.001 ) related their symptoms to physical rather than emotional problems . in response to the question about the cause of illness , people with rural cultural background used more also , women pointed to marital problems and men to life difficulties as the main causes of their illness ( p<0.001).despair prevailed among the singles ( p=0.012 ) . in general , this study confirmed our initial assumption about a different profile of depressive symptoms in iranians compared to the western population . our patients showed a high frequency of somatic symptoms , especially pain , as a manifestation of depression . contrary to the common findings discussed in the western literature , we could not find a difference between men and women in suicidal ideation and attempt . however , some of our findings are in accordance with the profile of depressive symptoms in western countries . because of the large number of the variables and to prevent confusion , the depressive symptoms are discussed as separate entities in the following sections . pain and physical symptoms this study showed the importance of somatization in a group of depressed patients who seemed to have no word for their emotions . , men have a dominant role and women often have no knowledge about their rights and their interests . our finding also confirmed the previous observation in that somatic symptoms played an important role in the manifestation of depressive disorder in iranian patients . the importance of somatic symptoms in the depressed patients has also been shown in many studies especially those performed in eastern countries . nieuwsma pointed out that social stigma is the main factor to complain of somatic symptoms instead of depression . fear of stigmatization and reluctance to appear as psychiatric patients are important factor for expressing their emotional pain via somatic route . the pattern of somatization , as kleinman noted in his study on chinese patients , may be unfamiliar to western clinicians and may further complicate the concept of depression . referral to psychiatrist the general practitioners and other specialists are still primary physicians for persons with lower education and people with rural cultural background . this can be related to the unavailability of psychiatrists in rural areas . however , the factor of knowledge has to be considered as a contributory parameter for evaluating this condition . the study conducted by bhui and colleagues revealed that south asians are more likely to visit their general practitioners and less likely to have a recognized mental disorder than white groups , and even if this is recognized , they are least likely to be referred to a specialist by gps . higher prevalence of guilt feeling in patients with urban cultural background is a finding comparable with the results of who collaborative study which showed the higher prevalence of this symptom in western societies . one study showed that guilt feelings could be found in patients with both pakistani and austrian cultures , regardless of age and sex . in fact , guilt feeling was associated with the severity of psychomotor retardation in depression . in some patients , , the idea of original sin rooted in the christianity teachings plays a major role in the guilt feeling of depressed patients . the lesser prevalence of guilt feeling in the middle aged group can be related to the social activity and the functionality of these people compared to the elders and younger people . hypochondriac ideation according to the dsm - iv , hypochondriasis is a disorder with relatively similar prevalence in men and women . accordingly , in this study we could not find a significant difference between men and women in hypochondriac ideations . hypochondriasisas is an obsession about death and it can be an explanation for the higher prevalence of this symptom in the elderly . similarity of the prevalence of hypochondriac ideations in rural and urban areas may indicate that this symptom is less affected by the cultural backgrounds of patients than somatization . as a vegetative symptom , insomnia is also more frequent in the lower educated people and in rural areas and is associated with somatic symptoms . logistic regression analysis also showed the interplay of insomnia and somatic symptoms ( table 4 ) . whether this is representative of sleep disturbance due to somatic problems or vice versa remains to be clarified . factors associated with the chief complaint of somatization like insomnia , this can be affected by somatic symptoms , especially gastrointestinal manifestations . however , because of the similarity of weight loss in the rural and urban areas , this symptom can be a subjective feeling rather than a true anorexia leading to weight loss . loss of concentration this condition has frequently been reported by younger patients who may be more concerned about their memory problems than older people . however , the elders deny their defects in memory and do not complain about it . suicidal ideation and attempt according to the dsm - iv , suicidal ideation is more frequent in women and suicidal attempt is more prevalent in men . however , our study showed no difference in suicidal ideation and attempt between men and women . suicidal idea was more frequent among rural inhabitants , but no difference in suicidal attempt was found between them and urban population . perhaps this may be related to the hesitation of rural people in expressing their emotions . our findings of higher prevalence of suicidal attempt and ideation in married patients is in contrast with those of other studies . also , our study showed that both suicidal ideation and attempt are more frequent in the younger age group . this findings is in conflict with some studies that consider the old age as a risk factor for suicidal attempt . this difference can be related to the supportive care giving received by the older members of the family in eastern countries like iran . logistic regression analysis showed the interplay between suicidal ideation and somatic symptoms , but not between suicidal attempt and hopelessness with somatic symptoms . the higher prevalence of despair in singles is suggestive of the seriousness of the problem in this group . although a cause and effect relationship can not be derived from a cross - sectional study , the symptoms of hypochondriasis , suicidal idea , crying , irritability and insomnia were significantly associated with the complaint of somatization ( table 4 ) . one possible explanation is that somatic problems of patients disrupt their sleep and increase their vulnerability to environmental and emotional stimuli . this in turn , can aggravate the patients affective stability presented by more crying , irritability or thoughts of death . on the other hand , sleeping disorders may also lead to somatic complaints . considering the covariant variables in the regression analysis , only gender was associated with the complaint of somatization in patients with major depressive disorder . however , neither of the educational level and patients age are associated with the complaint of somatization . these results indicated that somatization as a complaint in the patients with major depressive disorder is independent from age and educational level . one explanation could be that somatization as a complaint in iranian culture is commonly expressed in both higher and lower educated class . the principal finding of this study is that somatic symptoms especially headache and pain in other areas of the body have a significant weight in the chief complaints of depressed patients . therapists need to pay attention to the various ways of presentation of this disorder in different cultures in order to understand the symptoms of patients with depression . hence , more extensive studies in other areas of the country are required to obtain a more reliable profile of depression symptoms in iranian population . non - hodgkin lymphomas ( nhls ) account for approximately 60% of all lymphomas in children and adolescents . childhood nhls are subdivided into burkitt 's lymphoma , diffuse large b - cell lymphoma ( dlbcl ) , lymphoblastic lymphoma , and anaplastic large - cell lymphoma . dlbcl is characterized by relatively more frequent extranodal presentation , seen in upto 40% of the cases . primary involvement of the lymphoma of the middle ear is rare , with only about 18 cases being reported in literature so far . here , we report a case of dlbcl , presented with features of facial palsy and otitis , who received initial symptomatic treatment and later chemotherapy after diagnosing dlbcl . a 2 years 8 months old boy visited our tertiary care hospital with complaints of ear ache ( left side ) and left facial palsy of 4 weeks duration . after initial 2 weeks of these symptoms , there was whitish serous discharge from the left ear . before referral to our hospital , he was treated for otitis media with antibiotics , details of which were not available . his complete blood picture and biochemistry investigations done in our hospital were within normal limits . as the symptoms persisted for 4 weeks , computed tomography of head and neck was done which was suggestive of solid mass lesion of 2 cm 2 cm size in the left mastoid with destruction of mastoid bone [ figure 1 ] . the disease was in stage 1 ( as per murphy 's staging ) and was completely resected . histopathological examination showed large cells of lymphoid cell proliferation immune histochemistry was positive for cd20 , bcl-2 and negative for cd3 , with low mib-1 , which confirmed dlbcl . his positron emission tomography for staging , bone marrow and cerebrospinal fluid revealed no abnormality . computed tomography head and neck suggestive of solid mass lesion of 2 cm 2 cm size in the left mastoid with destruction of mastoid bone computed tomography head and neck of right side showing no abnormality his initial clinical symptoms of earache and facial nerve palsy followed by ear discharge resolved after starting chemotherapy as per b - cell lymphoma protocol for 6 months . currently , he is 37 months off treatment , and no disease recurrence is seen clinically as well as radiologically . it is an aggressive form of lymphoma , usually curable with appropriate treatment and has high survival rate . rapid disease progression of dlbcl calls for an early , accurate diagnosis and appropriate treatment . however , unusual presentation can mislead the physician resulting in wrong diagnosis , which delays the treatment , thus promoting disease progression . extranodal presentations of childhood dlbcl are relatively uncommon in clinical practice , and much rarer are those primarily involving middle ear and mastoid . involvement of middle ear and mastoid can resemble the features of otitis media and unusual facial palsy mimicking mastoiditis . although these initial symptoms were suggestive of middle ear infection , the distinguishing factor was unresponsiveness to antibiotics . there have been very few reports of nhl with facial nerve involvement ; ogawa et al . mccabe et al . , reported a case of 2-year - old , an immunocompetent boy with spontaneous regression of an epstein - barr - virus - associated monoclonal lymphoid proliferation who presented with acute otitis media and facial palsy . have described a case of dlbcl with features of otitis media , mastoiditis , and facial palsy . as symptoms persisted even after 4 weeks of treatment , the patient was started on chemotherapy , to which he promptly responded . extranodal nhls of middle ear which is not a common clinical presentation may present as facial palsy , and misdiagnosed as otomastoiditis . there should be a high index of suspicion for primary neoplasms of the middle ear in patients with chronic otomastoiditis refractory to appropriate initial antibiotic therapy ; early diagnosis and appropriate treatment results in good therapeutic outcome and minimizes further complications .

background : in some cultures , including ours , direct explanation of inner psychic world is inhibited and stigmatized , therefore finding alternative modes of expression . the aim of this cross - sectional study was to assess the frequency of somatization in the depressed patients . methods : the present study comprised 500 patients referred to the outpatient clinic of shiraz university of medical sciences , and diagnosed with major depressive disorders based on dsm - iv - tr . the presenting complaints of these patients were assessed through psychiatric interview . the presenting symptoms were divided into three main categories including mental symptoms , pain , and physical symptoms without pain . statistical analysis ( chi - square and logistic regression ) were performed to determine the relationship between presenting symptoms and some demographic variables such as age , gender , marital status , educational level and cultural background ( urban or rural ) . results : physical symptoms other than pain , mental symptoms , and pain were found in 193 ( 38.6% ) , 186 ( 37.2% ) , and in 121 ( 24.2% ) patients respectively . pain and physical complaints were more common in patients with rural cultural background , lower education , women and the married individuals . headache ( 15.2% ) , irritability ( 10.6% ) and pain in different parts of the body ( 10.4% ) were the most frequent chief complaints of the patients . hypochondriasis , suicidal idea , crying , irritability and insomnia were significant symptoms associated with the complaint of somatization . conclusion : somatic symptoms , especially pain , have a significant weight in the chief complaints of depressed patients . physicians need to pay particular attention to this important issue in order to better understand these patients . extra nodal presentation of non hodgkins lymphoma ( nhl ) is a rare entity , and data available about the nhl that primarily involves of middle ear and mastoid is limited . we report a case of diffuse large b cell lymphoma ( dlbcl ) , in a 2 year 8 month old boy , who developed otalgia and facial palsy . computed tomography revealed a mass in the left mastoid . mastoid exploration and histopathological examination revealed dlbcl . this case highlights the importance of considering malignant lymphoma as one of the differential diagnosis in persistent otitis media and / facial palsy .

the goal of these drug cocktail regimens is to achieve additive or synergistic effects among chemotherapeutics , thereby maximizing summation dose - intensity with resultant enhanced anticancer activities and increased patient survival . combinations have been identified and developed both through unbiased approaches and by rational design , and compounds that act on a single biochemical pathway are particularly strong candidates for synergy or potentiation . for example , inhibitors of poly(adp - ribose)polymerase-1 ( parp-1 ) , an enzyme that facilitates dna damage repair , potently synergize with dna damaging agents as demonstrated in cell culture and animal models . herein we describe an approach for potentiation not based on compounds acting on two targets within a single pathway , but rather with two compounds acting differentially to activate the same enzyme . during apoptosis , the zymogen procaspase-3 is activated via proteolysis to caspase-3 , and this active caspase-3 then cleaves scores of cellular substrates , executing the apoptotic program . as procaspase-3 protein levels are elevated in various tumor histologies , drug - mediated direct activation of procaspase-3 has gained significant interest as a selective anticancer strategy . furthermore , caspase-3 has been shown to play critical roles in cardiomyocyte hypertrophy , cellular differentiation , and remodeling . thus development of a strategy to magnify the timing and level of caspase-3 activation in a specific and direct manner could greatly aid the study of active caspase-3 in these nonapoptotic processes . to date , two major classes of compounds have been disclosed that enhance the activity and automaturation of procaspase-3 in vitro and induce apoptosis in cancer cells in culture . procaspase - activating compound-1 ( pac-1 , figure 1a ) enhances the activity of procaspase-3 in vitro via the chelation of inhibitory zinc ions , induces apoptosis in cancer cells in culture , and has shown efficacy in multiple murine tumor models . more recently , the compound 1541b ( figure 1a ) was discovered to promote the automaturation of procaspase-3 to caspase-3 in vitro and to induce apoptotic death of cancer cells in culture . compound 1541b appears to activate procaspase-3 via a binding - induced shift in the on off state equilibrium , or through formation of nanofibrils . pac-1 and 1541b exert their activating effect on procaspase-3 by distinct biochemical mechanisms , suggesting the potential for synergistic effects in cell culture and in vivo . ( b ) procaspase-3 ( 100 nm ) was incubated with pac-1 or 1541b in zinc - supplemented caspase activity buffer . data is normalized to procaspase-3 ( 100 nm ) activity in zinc - free caspase activity buffer as 100% activity . ( c ) procaspase-3 ( 350 nm ) in zinc - supplemented caspase activity buffer was incubated 1541b , pac-1 , or 1541b + pac-1 , and enzymatic activity was assessed with ac - devd - pna . data is normalized to 100% activity = 350 nm caspase-3 in zinc - free caspase activity buffer . error bars represent the sem of three replicates . in vitro , procaspase-3 has enzymatic activity that is dramatically lower than caspase-3 , with estimates ranging from reductions of 200 to 10 fold . low micromolar levels of zinc inhibit the activity of procaspase-3 and caspase-3 in vitro . pac-1 , a moderate affinity zinc chelator that has been shown to chelate the labile zinc pool in cells , allows procaspase-3 to once again process substrates , including itself . described herein is the combination use of the two small molecule activators of procaspase-3 , pac-1 and 1541b . these compounds act synergistically to enhance procaspase-3/caspase-3 activity in vitro , induce rapid procaspase-3 processing and caspase-3 activity in cell culture , potently and rapidly cause apoptotic death in a variety of cancer cell lines , and have efficacy in a murine tumor model . as zinc colocalizes with cellular procaspase-3/caspase-3 , it was of interest to determine if 1541b could activate recombinantly expressed procaspase-3 ( supporting figure s1a ) in the presence of zinc ; the activating effect of 1541b on procaspase-3 in vitro had previously only been evaluated under zinc - free conditions . as shown in figure 1b , compound 1541b does not enhance intrinsic procaspase-3 enzymatic activity in zinc - containing buffers , whereas pac-1 relieves zinc - mediated inhibition and induces the anticipated activating effect . this effect from pac-1 is reliant upon its zinc binding ability , as an analogue that does not bind zinc ( pac-1a ) is not active in this experiment . in addition , an inactive derivative of 1541b ( compound 1541d ) has no effect in this experiment ( structures and data in supporting figure s1b d ) . this orthogonal activity of pac-1 and 1541b on procaspase-3 ( pac-1 activating zinc - bound procaspase-3 , 1541b activating procaspase-3 with zinc removed ) suggests synergistic potential , and indeed , the combination of pac-1 and 1541b , when incubated with procaspase-3 in vitro , leads to dramatic caspase-3 activity far exceeding the effect of either compound alone ( figure 1c ) . consistent with the established structure activity relationships , pac-1a and 1541d as single agents and in combination were unable to facilitate procaspase-3 activation in vitro ( supporting figure s1d ) . to examine pac-1 synergy with 1541b for activation of procaspase-3 in cancer cells in culture , a panel of cancer cell lines were treated with combinations of pac-1 and 1541b and the caspase-3/-7 activity of the cell lysates was monitored with the fluorogenic caspase substrate ac - devd - afc . as seen in figure 2a , cotreatment of u-937 ( human lymphoma ) , bt-549 ( human breast cancer ) , and a549 ( human lung cancer ) cells with pac-1 and 1541b results in markedly more dramatic and rapid increases in devdase activity than treatment with either pac-1 or 1541b as single agents . the combination treatment stimulates caspase activity that rivals or surpasses that induced by staurosporine ( sts , 1 m ) , one of the most rapid inducers of cellular devdase activity known , and is dose dependent ( supporting figure s2 ) . pac-1/1541b combinations induce rapid and dramatic procaspase-3 activation and maturation in cancer cell lines in culture . ( a ) cancer cell lines were treated with pac-1 ( 30 m ) , 1541b ( 15 m ) , or the combination , cells were lysed at various time points , and caspase-3/-7 activity of the lysates was evaluated with the fluorogenic ac - devd - afc substrate . ( b ) western blot of various cancer cell lines after treatment with combinations of pac-1 ( 30 m ) and 1541b . treatment durations of 3 , 4 , and 8 h for u-937 , bt-549 , and a549 cells , respectively , were chosen on the basis of the timing of maximal caspase activity , as observed in cell lysate . to determine if the elevation of devdase activity was the result of enhanced cleavage of procaspase-3 to caspase-3 facilitated by compound cotreatment , cells were treated with pac-1 and 1541b combinations and assessed by western blotting . as shown in figure 2b , dramatic activation of procaspase-3 to caspase-3 was observed in u-937 , bt-549 , and a549 cells upon treatment with the pac-1/1541b combinations , whereas low / no procaspase-3 activation was observed with 1541b or pac-1 alone at the times and concentrations evaluated . analogous results in hl-60 ( human leukemia ) , hs578 t ( human breast cancer ) , and el4 ( murine lymphoma ) cells are shown in supporting figure s3 . apoptosis through the intrinsic pathway proceeds via an ordered series of events , with bcl-2 family proteins acting on the mitochondria , stimulating cytochrome c release , formation of the apoptosome , caspase-9 activation , and subsequent cleavage of procaspase-3 to caspase-3 . caspase-3 then cleaves scores of substrates , executing the apoptotic program . to examine these events in cells treated with pac-1 + 1541b , bt-549 cells were assessed for the timing of procaspase-3 activation relative to cytochrome c release from the mitochondria by western blot of fractionated cell lysate . as shown in figure 3a , analysis of cells treated with pac-1 + 1541b shows the appearance of cleaved caspase-3 bands in the cytosolic fraction before cytochrome c levels diminish in the mitochondria . at these compound concentrations and relatively short treatment times , 1541b as a single agent is considerably less effective at inducing procaspase-3 activation ( figure 3b ) , and pac-1 is not effective . importantly , as shown in figure 3c , staurosporine induces apoptosis through the canonical intrinsic pathway , with clear release of cytochrome c from the mitochondria prior to activation of procaspase-3 . rapid procaspase-3 activation is observed in cells treated with pac-1 + 1541b , occurring prior to release of cytochrome c from the mitochondria . ( a ) bt-549 breast cancer cells were treated with pac-1 ( 30 m ) and 1541b ( 10 m ) over 8 h and assessed in the cytosol for procaspase-3 activation , and in the mitochondria for cytochrome c levels . ( b ) bt-549 cells treated with 1541b ( 10 m ) and assessed for 8 h. ( c ) bt-549 cells treated with sts ( 1 m ) and assessed for 10 h. bt-549 cells were assessed by phase contrast microscopy and monitored for the phenotypic changes associated with caspase activation and apoptosis ( supporting figure s4 ) . consistent with the western blot and caspase activity data , only the combination of pac-1 and 1541b demonstrated substantial morphological changes indicative of apoptosis , such as blebbing . the combination of pac-1 and 1541b was evaluated for the capacity to induce apoptotic death in a variety of cancer cell lines in culture . these evaluations were performed at short incubation times , reflective of the timing of caspase activation observed in figure 2 and where neither compound exerts a significant effect as a single agent . pac-1 significantly synergizes with 1541b for potent proapoptotic activity in u-937 , bt-549 , and a549 cells ( figure 4a ) , and in hl-60 , hs578 t , u-87 , and el4 cells ( supporting figure s5 ; see supporting figure s6 for representative annexin v - fitc / propidium iodide histograms ) ; the dashed horizontal lines in each graph mark levels of cell death that would be observed from a strictly additive effect of pac-1 and 1541b . while the non - blood - brain barrier permeable analogue s - pac-1 possesses a comparable capacity for synergy as pac-1 ( supporting figure s7 ) , synergy was not observed when either pac-1a or 1541d were used in combination with 1541b or pac-1 ( supporting figures s8 and s9 ) . pac-1 and 1541b synergize to induce rapid cell death in cancer cell lines . ( a ) cancer cell lines were treated with the indicated concentrations of pac-1 ( 0 , 15 , and 30 m ) and 1541b ( 0 , 7.5 , 10 m ) for six hours , and apoptotic death was assessed using flow cytometry with annexin v - fitc / propidium iodide staining . the dotted horizontal lines represent the level of cell death expected from a mere additive effect of pac-1 and 1541b for each drug combination . ( b ) percent cell death observed after 6 h treatment of u-937 cells with pac-1 + 1541b combinations in matrix format . values are heat mapped with white equal to 0% cell death and red equal to 100% cell death . ( c ) combination index values calculated for each combination with combosyn software ( < 1 indicates synergistic interaction with values < 0.3 indicating strong synergism ) . values are heat mapped with lowest values in green and highest values in red . further assessment ( vs u-937 cells ) of a broad range of concentrations for both compounds clearly shows the dramatic synergy between these two agents . shown in figure 4b is the cell death induced by the compound matrix , with synergy apparent from simple comparisons of the percent cell death induced by the single agents versus the combinations ; this is true at both short ( 6 h , figure 4b ) and long ( 24 h , supporting figure s10 ) time points . synergism is frequently quantified in such experiments though calculation of combination indices ( ci ) . combinations describing synergistic interactions possess ci values < 1 , while antagonistic interactions have ci value > 1 ; additive effects occur when the ci value equals 1 . when u-937 cells were treated with a range of pac-1 and 1541b compound concentrations in combination , synergistic interactions were observed broadly , with several combinations considered strongly synergistic ( figure 4b and c and supporting figure s10 ) . the proapoptotic effect of the pac-1/1541b combination was markedly reduced with the pan - caspase inhibitor q - vd - oph , consistent with the involvement of caspases in the mode of cell death ( figure 5a ) . to further investigate the connection between activation of procaspase-3 and the cell death induced by the drug combination , mcf-7 cells were used , a cell line that does not express procaspase-3 , together with a matched mcf-7 cell line where procaspase-3 is expressed via a plasmid ( figure 5b ) . the combination of pac-1/1541b has minimal effect on mcf-7 cells transfected with control plasmid ( mcf-7 vrl ) , but dramatically induces apoptosis in procaspase-3 expressing mcf-7 cells ( mcf-7 c-3 ) ( figure 5c ) , an effect correlated with procaspase-3 activation ( figure 5d ) . pac-1 and 1541b induce rapid caspase - dependent cell death in cancer cell lines . ( a ) the pan - caspase inhibitor q - vd - oph ( 25 m ) protects against pac-1/1541b - mediated cell death in u-937 cells . ( b ) western blot analysis of mcf-7 cells transformed with empty plasmid ( mcf-7 vrl ) and mcf-7 cells transformed with a plasmid containing the gene for procaspase-3 ( mcf-7 c-3 ) . ( c ) mcf-7 vrl and mcf-7 c-3 cells were treated with the indicated concentrations of pac-1 and 1541b and apoptotic death was assessed using flow cytometry with annexin v - fitc / propidium iodide staining . the dotted horizontal lines represent the level of cell death expected from a mere additive effect of pac-1 and 1541b for each drug combination . ( d ) western blot of mcf-7 c-3 cells treated for 6 h with combinations of pac-1 ( 30 m ) and 1541b . ( e ) the combination of pac-1 and 1541b has an antitumor effect in vivo . el4 cells were injected subcutaneously into c57bl/6 mice ( 10 million cells per mouse ) , the animals were split into four groups and treated once - a - day for three days via ip injection with vehicle ( 2-hydroxypropyl--cyclodextrin , hpcd ) , 1541b ( 17.5 mg / kg in hpcd ) , pac-1 ( 125 mg / kg in hpcd ) , or 1541b + pac-1 ( 17.5 and 125 mg / kg , respectively , in hpcd ) . p - values are relative to vehicle control ; * , p < 0.005 ; * * , p < 5 10 . to explore the therapeutic utility of the dual procaspase-3 activation strategy , the combination of pac-1 and 1541b was assessed in vivo . prior to beginning efficacy studies , the tolerability and toxicity of 1541b as a single agent and in combination with pac-1 was determined ( supporting tables s1 , s2 , and s3 ) . on the basis of these results , a maximum combination dosage of 125 mg / kg of pac-1 followed by 17.5 mg / kg of 1541b was chosen for the efficacy model . the hematologic and nonhematologic toxicity of this treatment of pac-1 and 1541b was determined following 3 consecutive daily ip administrations of hpcd , pac-1 , 1541b , or combination ( pac-1 + 1541b ) . no clinically significant evidence for myelosuppression , renal injury , or hepatic toxicity was identified in any of the treatment cohorts ( supporting table s3 ) . for mice receiving hpcd , pac-1 , or 1541b alone , all collected target organs were devoid of histologic evidence for inflammation and necrosis . in mice receiving combination ip pac-1 and 1541b , pathologic changes were noted in the lung parenchyma of 2 out of 3 mice . these mice had eosinophilic and histiocytic perivascular and peribronchiolar infiltrates , which were mild to moderate in severity . the clinical significance of this finding is unclear , as mice in these groups were asymptomatic and did not demonstrate weight loss compared to controls . the pharmacokinetic parameters of each compound were then determined ( supporting table s4 ) . following a single ip injection at a dose of 125 mg / kg , pac-1 in hpcd was rapidly absorbed and achieved a maximal plasma concentration of 32720.0 ng / ml within 20 min postinjection . pac-1 was distributed quickly throughout the body with a mean distribution half - life of 20 min and a mean terminal elimination half - life of 4.2 h. likewise , 1541b in hpcd was absorbed and reached a maximal plasma concentration of 2699.9 ng / ml within 20 min of intraperitoneal administration . the distribution half - life of 1541b was 20 min and the terminal elimination half - life was 5.4 h. the el4 syngeneic murine lymphoma model was chosen to explore the therapeutic utility of pac-1 and 1541b combinations , as pac-1 and 1541b synergize to induce dramatic activation of procaspase-3 and cell death versus this cell line ( supporting figures s3c and s5d ) , and it is a challenging treatment model due to its rapid doubling time ( less than 24 h ) and uniformly high tumor formation rate ( 100% ) when inoculating greater than 1 10 cells subcutaneously in c57bl/6 mice . pac-1 was chosen over s - pac-1 for the combination studies as it has greater activity versus the el4 cell line in culture . c57bl/6 mice implanted with el4 cells were treated with pac-1 alone ( 125 mg / kg ) , 1541b alone ( 17.5 mg / kg ) , or sequential treatments of pac-1 + 1541b ( 125 mg / kg and 17.5 mg / kg , respectively ) once - a - day for three days , formulated in hpcd . once tumors in vehicle - treated mice had reached their maximum allowed size of 1500 mm ( 8 days ) , all mice were sacrificed , and tumors were excised and weighed . as shown in figure 5e , 1541b treatment had no effect , and pac-1 had a small but statistically significant effect on tumor growth in this model . while there is clear benefit to anticancer strategies utilizing combinations of drugs that act on different targets , the work described herein demonstrates that dramatic synergy can be observed with compounds that act through orthogonal mechanisms on the same biological target . this multitargeting approach may have particular advantages when activation of an enzyme is sought . pac-1 chelates the labile inhibitory zinc from procaspase-3/caspase-3 , thus priming the enzyme for robust and efficient activation by 1541b . in cell culture at concentrations where neither compound significantly induces death at 612 h , dramatic enhancement of cell death ( over the additive effect ) is observed with the pac-1/1541b combination . this cell death is tied to the ability of the pac-1/1541b combination to induce a rapid conversion of procaspase-3 to caspase-3 , as shown by the western blots and the caspase-3 enzymatic activity in cell lysates . pac-1 is safe in mice and dogs , and a derivative of pac-1 , s - pac-1 , showed promising activity in a phase i clinical trial of pet dogs with lymphoma , thus the observed synergy with 1541b could have clinical significance . pac-1 has been utilized as a tool for studying direct procaspase-3 activation in various systems , and data presented herein suggest that the pac-1/1541b combination will also be valuable as a tool for rapidly activating procaspase-3 . as interest in activating enzymes with small molecules is increasing rapidly and has considerable medicinal potential , our data suggest that targeting strategies using two small molecules with different activation mechanisms could be a general approach for dramatic enhancement of the intended biological effect . procaspase-3 and caspase-3 were expressed as described previously and purified with qiagen nickel - nta resin . increasing concentrations of pac-1 , 1541b , pac-1a and 1541d were assessed for their capacity to enhance activity of 100 nm procaspase-3 in an inhibitory zinc - based system ( 2 m znso4 , 50 mm hepes , 50 mm kcl , 1.5 mm tcep , 0.1% chaps , ph 7.6 ) . compounds were added to zinc - inhibited pc-3 , incubated for 30 min , and activity was assessed by cleavage of ac - devd - afc ( 50 m ) and was normalized to 100 nm procaspase-3 in zinc - free caspase activity buffer ( 50 mm hepes , 50 mm kcl , 1.5 mm tcep , 0.1% chaps , ph 7.6 ) . 1541b ( 20 and 25 m ) and pac-1 ( 25 and 50 m ) were evaluated separately and in combination for their capacity to activate 350 nm procaspase-3 over time in a mildly inhibitory zinc - based system ( 750 nm znso4 , 50 mm hepes , 50 mm kcl , 5 mm tcep , ph 7.6 , 0.1% tritonx-100 ) . at designated time points aliquots were assessed by cleavage of ac - devd - pna ( 200 m ) . 350 nm caspase-3 in a zinc - free caspase activity buffer ( 50 mm hepes , 50 mm kcl , 5 mm tcep , ph 7.6 , 0.1% tritonx-100 ) was used for 100% caspase-3 activity . u-937 ( 50 000 cells per well ) , bt-549 ( 10 000 cells per well ) and a549 ( 10 000 cells per well ) were plated in 96-well plates , allowed to adhere overnight ( bt-549 and a549 ) , and incubated with 1 m staurosporine ( sts ) , 1541b ( 7.5 , 10 , 12.5 , or 15 m ) , pac-1 ( 30 m ) , 1541b ( 7.5 , 10 , 12.5 , or 15 m ) and pac-1 ( 30 m ) or dmso ( final concentration 1% ) in phenol - red free rpmi media . plates were assessed for executioner caspase activity via addition of a 4 bifunctional lysis activity buffer ( 200 mm hepes , 400 mm nacl , 40 mm dtt , 0.4 mm edta , 1% tritonx-100 , 20 m ac - devd - afc ) . activity is expressed as normalized to minimal and maximal activity observed within the assay . for western analysis one million suspension ( u-937 , hl-60 and el4 ) or adherent cells at 75% confluency in 6-well plates were treated with increasing concentrations from 7.5 to 12.5 m 1541b in the presence or absence of 30 m pac-1 for a duration reflective of near maximal caspase activity as seen in the cell lysate experiment . at the conclusion of treatment , the medium and trypsin - aided detached cells were pelleted , lysed on ice in ripa buffer ( 50 mm tris base , 150 mm nacl , 1% tritonx-100 , 0.5% na - deoxycholate , 0.1% sds , ph7.4 , with a 1:100 dilution of protease inhibitor cocktail set iii ) , and clarified , and protein content was normalized by bca protein assay reagent ( pierce ) . samples were denatured , separated by sds - page ( 420% ) , and transferred to a membrane for western blot analysis of pro- and active caspase-3 ( cell signaling 9662 ) . blots were stripped and reprobed for -actin ( cell signaling 4970 ) as a loading control . bt-549 cells at 75% confluency ( allowed to adhere for > 24 h ) in 6-well plates were treated with 10 m 1541b and 30 m pac-1 , 10 m 1541b or 1 m sts for up to 10 h. at each time point , medium and trypsin - aided detached cells were pelleted , washed in pbs and suspended in cold digitonin permeabilization buffer ( 75 mm nacl , 1 mm sodium phosphate monobasic , 8 mm sodium phosphate dibasic , 250 mm sucrose , 200 g / ml of digitonin , protease cocktail inhibitor , ph 7.5 ) and placed on ice for 5 min . permeabilized cells were centrifuged ( 14 000 rcf , 5 min ) , and the supernatant ( cytosolic fraction ) was saved . the pellet ( mitochondrial fraction ) was washed with digitonin permeabilization buffer , lysed in ripa buffer , and clarified . samples were denatured , separated by sds - page and transferred to a membrane for western blot analysis probed for the presence of pro- and active caspase-3 ( cell signaling 9662 ) , and cytochrome c ( cell signaling 4272 ) . western blots were stripped and reprobed for -actin ( cell signaling 4970 ) and cox iv ( cell signaling 5247 ) as loading controls . the induction of apoptosis was measured by annexin v - fitc / propidium iodide staining and flow cytometry . either 1 000 000 suspension ( u-937 , hl-60 , el4 ) cells or adherent cells at 75% confluency in 6-well plates were treated with combinations of pac-1 and 1541b ( final concentration of dmso the entire contents of each well was then transferred to flow cytometry tubes , pelleted , and suspended in 450 l of annexin v - fitc binding buffer ( 10 mm hepes , 140 mm nacl , 2.5 mm cacl2 , 1% bsa , ph 7.4 ) , premixed with annexin v - fitc and propidium iodide dyes . staining was assessed by flow cytometry ( 10 000 events per sample ) . the annexin v - fitc ( )/propidium iodide ( ) population was confirmed to be greater than 90% viable and normalized as the live control . the role of caspases in the induction of apoptosis was assessed by cotreatment with 25 m q - vd - oph ( calbiochem ) . all experimental procedures were reviewed and approved by the university of illinois institutional animal care and use committee . 68 week old c57bl/6 mice were used in all experiments ( charles river ) . the maximum tolerated dose ( mtd ) of 1541b was determined . beginning at 5 mg / kg , ( formulated at 0.5 mg / ml in hpcd , ph 5.5 ) mice ( groups of three ) mtd was the highest dosage for which all mice survived and acceptable ( < 20% ) weight loss was observed . the mtds of pac-1 ( 200 mg / kg ) and 1541b ( 20 mg / kg ) were used as the base unit for determining the combined mtd . beginning with 1/10 of each mtd ( 20 mg / kg pac-1 + 2 mg / kg 1541b ) groups of 3 mice were treated with 1/10 , 1/6 , 1/4 , 1/3 , 1/2 and 3/4 of each single agent s mtd . given the tolerability of 1/2 and the toxicity of 3/4 , an additional treatment of 125 mg / kg pac-1 + 17.5 ten week old , c57bl/6 female mice ( n = 3/cohort ) were administered three consecutive daily intraperitoneal injections of hydroxypropyl--cyclodextrin ( vehicle ) , pac-1 ( 125 mg / kg ) , 1541b ( 17.5 mg / kg ) , or pac-1 + 1541b ( 125 mg / kg + 17.5 mg / kg , respectively ) , and then humanely sacrificed 24 h later . heparinized whole blood was collected for assessment of total white blood cells , neutrophils , lymphocytes , hematocrit , platelets , creatinine , blood urea nitrogen , albumin , alanine aminotransferase , alkaline phosphatase , and total bilirubin . mice were necropsied , and heart , lung , kidney , liver , spleen , gastrointestinal tract and brain were collected for histopathology . tissue samples were fixed overnight in 10% neutral buffered formalin , processed , and paraffin embedded for histopathology using routine methods . tissue blocks were sectioned into 3 m tissue sections and stained with hematoxylin and eosin . all slides were systematically evaluated by a single board certified veterinary pathologist ( lbb ) for evidence of acute or chronic inflammation and toxicity . all lesions were characterized , recorded , and scored for severity ( minimal = 1 , mild = 2 , moderate = 3 , and severe = 4 ) . ten week old , c57bl/6 female mice were administered a single intraperitoneal dose of pac-1 at 125 mg / kg or 1541b at 17.5 mg / kg , and sacrificed in cohorts of 3 at predetermined time points ( 0 , 10 , 20 , 30 , 40 , 60 , 120 , 240 , 360 , 720 , and 1440 min ) . blood was collected and centrifuged , and plasma separated for quantification of pac-1 or 1541b by hplc methods ( uiuc metabolomics center , urbana , il ) . pharmacokinetic analyses were performed , using a nonlinear regression program ( winnonlin , version 5.1 , pharsight corporation , cary , nc ) . ten million el4 cells were prepared in hbss and injected subcutaneously on the right flank of sedated ( ketamine / xylazine ) mice ( day 0 ) . by three hours postinjection the injection bleb was no longer evident , and soft tumors appeared the following morning . mice were randomized into four treatment groups : vehicle , 1541b alone , pac-1 alone , and 1541b + pac-1 . compounds were formulated in hpcd ( 1541b at 0.5 mg / ml at ph 5.5 and pac-1 at 12.5 mg / ml at ph 5.5 ) . all mice received two treatments , one hour apart : vehicle ( hpcd + hpcd ) , 1541b ( hpcd + 1541b ) , pac-1 ( pac-1 + hpcd ) and 1541b + pac-1 ( pac-1 + 1541b ) . after 8 days the largest tumors had achieved maximal size ; mice were sacrificed , and tumors were excised and weighed . diabetic retinopathy is the name given to the changes in the retina , which develop over a period of time in diabetics . it remains one of the major causes of new - onset visual loss in developed countries . if the central part of the retina ( i.e. , the macula ) is involved , it is referred to as diabetic maculopathy . the traditional approach to diagnosis of diabetic maculopathy includes fundus ophthalmoscopy and fluorescein angiography ( fa ) . the early treatment diabetic retinopathy study ( etdrs ) identified stereoscopic slit - lamp biomicroscopy and stereo colour fundus photography as standard methods of macular thickness assessment utilized in order to determine whether the treatment should be commenced as they defined the clinically significant macular oedema ( etdrs report number 10 , 1991 ) . however , these methods are subjective and relatively insensitive to small changes in retinal thickness and , therefore , may be unable to identify mild or localized macular thickening . they also do not provide any data on retinal morphology and blood flow . on the other hand , fa is a highly effective test of evaluating retinal blood vessels , macular perfusion , and pattern of leakage causing the oedema . although very useful clinically , it also does not contribute much to the evaluation of retinal morphology and its thickness profile . in 1991 the researchers from massachusetts institute of technology and harvard university patented the technique of optical coherence tomography ( oct ) , which was a major breakthrough in ophthalmic diagnostics ( us5321501 a , swanson ea , huang d , fujimoto jg , puliafito ca , lin cp , schuman js . method and apparatus for optical imaging with means for controlling the longitudinal range of the sample ) . the first paper to present the potential of the new diagnostic method four years later the first paper was published which described the use of oct in diagnosis of macular diseases . the purpose of this paper is to provide an overview of clinical utility of oct in retinal assessment of diabetic patients . oct enables obtaining the high resolution ( few micrometres ) cross - sectional images ( tomograms ) of the human retina in a noninvasive manner . in contrast to classic fundus photography taken with fundus camera , oct also provides information on the depth that the scattered light comes from . if light is reflected by the deeper retinal layers , it has to go a longer way to return to the detector compared to the light reflected from more superficial layers . this feature makes it possible to precisely determine what retinal depth ( i.e. , layer ) the particular signal comes from . therefore , oct resembles ultrasound imaging with the only difference consisting in utilizing light instead of sound . the use of light gives oct higher axial resolution compared to any other imaging techniques currently used in clinical medicine . in classic oct setup the light emitted by the superluminescent diode is directed to the beam splitter which splits it into two equal beams . one of them is projected onto the reference mirror , the other one onto the retina , and is backscattered from its morphological elements . the wave interference may occur only when the optical path between the beam splitter and the mirror is equal to the distance between the splitter and one of the surfaces reflecting the light within the retina . in that case , the detector will record the change in the light intensity . in order to detect other reflecting surfaces , the position of the reference mirror is referred to as time domain oct ( tdoct ) due to the fact that the information on retinal morphology along the scanning beam is obtained by recording the optical signal when the mirror is moved . this technique required the reference arm to be held fixed , and the optical path length difference between sample and reference reflections is encoded by the frequency of the interferometric fringes as a function of the source spectrum . the first is spectral domain oct ( sdoct ) in which interferometric signal is detected using the spectrometer equipped with a line of light sensitive elements . the other method is a swept source oct ( ssoct ) utilizing swept tunable lasers and a standard photodiode detector . as in fdoct , the reference mirror remains fixed , the better mechanical stability of the system is achieved . additionally , the interferometric signal created by mixing the sample and reference light is sampled as a function of wavenumber and yields an entire depth scan at the same time . this makes it possible to achieve several hundred - fold increase in speed and sensitivity of scanning compared to tdoct . as a result , significant motion artefacts are avoided and multiple measurements can be taken in a short time enabling the three - dimensional retinal scanning . the oct images can be also acquired at the video - rate and the measured structures observed in real time . the first commercially available sdoct device ( copernicus , optopol sa , poland ) the term reflectivity is used in oct technique as an equivalent of echogenicity in ultrasonography . the areas showing reduced reflectivity are referred to as hyporeflective , whereas the increased reflectivity regions are referred to as hyperreflective . the reflectivity in a grey - scale is proportional to tissue brightness observed in oct . the higher the reflectivity is , the closer to white the colour will be . in order to effectively visualise subtle structures in oct scans , the false colour scale is often clinically used , in which the individual colours are purely conventional . usually white and red represent highest intensity signal , whereas black and blue correspond to the lowest intensity signal . however , such approach has a disadvantage , namely , possible occurrence of artefacts . if signal intensity changes , the colour of a given structure on oct scan may also change . the oct results are presented as an axial scan , referred to as an a - scan , similarly like in ultrasonography . it is acquired by presenting the amplitude of the back - scattered light as a function of echo delay time . as the scanning beam moves along the retina , many a - scans are acquired , which form the tomogram , that is a b - scan ( figure 1 ) . it presents the cross section of the retina in a plane perpendicular to its surface . the set of many consecutive b - scans is assembled into a 3d reconstruction of retinal structure ( figure 2 ) . the software in - built in commercially available oct devices makes it possible to carry out a quantitative data analysis . the results of such analyses primarily include total retinal and individual layers thickness maps as well as macular volume maps . retinal thickness is usually calculated for central fixation point , 9 etdrs - like macular regions , and total macular thickness ( figure 3 ) . retinal volume is displayed for 9 etdrs - like macular areas and total macular volume . the oct data is automatically segmented in order to generate the above maps ( figure 2 ) . when interpreting these maps , one should bear in mind that the artefacts may occur during segmentation , which will lead to improper retinal thickness measurements [ 10 , 11 ] . artefacts may arise as a result of poor image quality , eye movement during measurements , and retinal pathologies interfering with automated segmentation ( e.g. , retinal pigment epithelial detachment , subretinal fluid , fibrosis , or haemorrhage ) . oct maps may be compared to normative data , including age , sex , and race ( figure 3 ) [ 1214 ] . it should be noted that different oct devices have different in - built normative databases . therefore , direct comparisons of maps generated by different oct devices are pointless [ 15 , 16 ] . the quantitative monitoring of retinal thickness in a given patient requires using the same oct device model for all follow - up examinations . it is quite intuitive , but prior to discussing the aspects of the retinal architecture seen in oct , the physical foundations of the obtained images should be explained . that is why the oct images should not be interpreted indiscriminately as histologic specimens . in histopathological examination , different stains show the affinity to different morphological elements , for example , cell nuclei . on the other hand , particular colours on oct cross - sectional image correspond to different signal intensity levels . however , since retinal oct images highly resemble histologic specimens , despite the above difference , and oct is a noncontact examination , it can be seen as an optical biopsy , which does not require tissue excision in order to analyse its structure . histologically , the retina consists of 10 layers , 4 of them being cellular and 2 neuronal junctions . the axonal layers , that is , the nerve fibre layer and the plexiform layers , are capable of potent light scatter and appear yellow to red on false - colour oct images . the light scattering potential of nuclear layers is lower , so they are represented as blue and black areas . the first layer visible on oct images is the internal limiting membrane which appears as a hyperreflective line at the vitreoretinal interface . it is visualized in oct owing to the increased light scatter between the transparent vitreous and retinal surface . below , there lies the retinal nerve fibre layer , typically thicker in the nasal macula and capable of potent light scatter . subsequently , hyperreflective plexiform layers are imaged as well as the inner nuclear layer situated between them , which has a lower light scattering potential . then , the relatively thick hyporeflective outer nuclear layer is visible with a thin hyperreflective line underneath . a distinct bright stria that stretched in front of the rpe demarcates the junction between the inner and outer photoreceptor segments ( is / os ) . due to the increased length of outer cone segments in central fovea it contains melanin and is capable of very potent light scattering . on the other hand , below , choriocapilaries and the rest of the choroid are visible . in the centre of the macula reduced reflectivity is most often cause by intraretinal and subretinal fluid accumulation ( oedema , retinal detachment , serous rpe detachment ) . pathological features that can be hyperreflective are : hard exudates , calcification , epiretinal and thick vitreous membranes , fibrosis , haemorrhages , rpe hyperplasia , neovascular membranes , atrophy of the retina and rpe causing increased reflectivity of underlying choroid . thus , it facilitates detecting macular oedema which is the main pathologic feature of diabetic maculopathy . this is defined as any detectable retinal thickening due to fluid accumulation ( etdrs report number 10 , 1991 ) . the oedema may be symmetrical or involve only a sector of the macular area . it usually starts as a focal lesion and progresses towards a more diffuse form . in some cases , the macular edges may be thickened , even though the contour of the foveal centre remains normal . persistent retinal oedema resulting in muller cell necrosis leads to the formation of cystoid cavities , located mainly in the outer retina ( henle 's fibre and outer plexiform layer ) , and sometimes also in the inner plexiform layer . in the most advanced stages in eyes with well - established long term macular oedema , several central cysts can merge together forming large hyporeflective cavity which contributes to the significant thickening of the fovea ( figure 4 ) . therefore , the main characteristics of macular oedema in oct , apart from increased retinal thickness , include intraretinal spaces of reduced reflectivity , disintegration of the layered retinal structure , and usually also flattening of the central foveal depression . in some cases fluid can be seen under the neurosensory retina ( figures 4 and 5(a ) ) . they present as small hyperreflective deposits with posterior shadowing ( figures 5(c ) and 5(d ) ) . that is why koleva - gorgieva proposed the classification of cystoid diabetic macular oedema ( dme ) into mild , moderate , and severe according to the size of cystoid spaces . the cystoid spaces in eyes with intermediate and severe cystoid dme are mainly located in the outer layers , predominantly in the fovea . in some cases small cysts in the inner layers can also be found . if the cysts continue to increase , they may occupy the full thickness of the retina , leading to its atrophy and the profound vision loss . in the past few years , since the introduction of fdoct , it has become possible to accurately visualize the outer retinal layers . several authors have reported that the integrity of elm and is / os junction has a positive correlation with visual acuity [ 21 , 22 ] . shin et al . showed that the photoreceptor layer status is closely associated with final visual acuity in dme and that photoreceptor integrity prior to treatment can be predictive of potential visual recovery in dme . yohannan et al . demonstrated that disruption of is / os junction correlates well with a significant decrease in point sensitivity in eyes with dme . therefore , the assessment of outer retinal layer structure should be a part of a routine evaluation when performing oct in patients with dme . the ability to visualize the vitreoretinal interface ( figures 6 , 7 , and 8) is a unique feature of oct . it allows for macular traction imaging , which may play a role in dme development [ 25 , 26 ] . the traction may be induced by vitreoretinal interface abnormalities such as incomplete posterior vitreous detachment ( pvd ) or epiretinal membrane ( erm ) . if the posterior hyaloid is thin and only slightly detached from the surface of the macula , it is not visible in ophthalmoscopy , but can be easily detected by oct . the same is true for erm ; if it is thin and does not cause a significant retinal distortion , it can be only visualized using oct . that is why the assessment of vitreoretinal interface is an essential step in macular evaluation in patients with diabetic retinopathy . moreover , oct does not only work well as a diagnostic tool in macular traction but may also be used in order to monitor the postoperative morphological outcomes ( figure 9 ) . it can also help identify the postoperative complications of vitrectomy , such as retinal detachment , erm , and lamellar macular hole formation . the detached posterior vitreous face presents oct scans as a thin hyper - to - medium reflective horizontal or oblique line in the non - reflective vitreous cavity , above or inserting into the retina . in case of incomplete pvd it can lead to increase in macular thickness , loss of foveal depression , and formation of intraretinal cystoid spaces or pseudoholes ( figure 6 ) . the distinction between erms and a pvd is usually made on the basis of reflectivity . oct can also be used to document the opacity and thickness of erm , its distance from the surface of the retina , and such effect on the underlying retina as distortion , oedema , or neurosensory detachment . it should be noted that oct is complementary to ultrasound scanning in evaluation of vitreoretinal interface . ultrasound scans provide a more complete image of vitreous pathology but at the cost of lower resolution . fdoct , on the other hand , provides a more detailed image of vitreoretinal interface but limited to a relatively small area ( figure 7 ) . as oct uses light to acquire the images , if the optic media are opacified and fundus can not be visualized , the retinal cross sections will not be obtained . the limitations are similar to the ones associated with ophthalmoscopy and fa ( figure 10 ) . however , sometimes oct images can be acquired in cases where retinal assessment in ophthalmoscopy is impossible ( figure 11 ) . in some cases oct can even enable assessment of the space located posteriorly to the thin fibrovascular membrane in proliferative retinopathy ( figure 12 ) . another particular value of oct is the possibility of reliable and reproducible retinal thickness measurements ( figure 5 ) . using the retinal thickness maps , it is possible to monitor dme progression and assess treatment outcomes after laser photocoagulation , intravitreal injections of anti - vegf and steroids or , as mentioned before , vitrectomy . owing to retinal thickness maps not only oedema but also atrophy can be detected , which contributes to lack of improvement or even decreased vision after the oedema is resolved ( figure 3 ) . the treatment efficacy in dme should , then , be evaluated in terms of two outcomes : the functional one based on visual acuity measurements and anatomical one assessed in oct . the first oct classification of dme presented by otani et al . was based on retinal morphological changes : sponge - like swelling , cystoid oedema , and serous retinal detachment . along with the improving oct technology , it takes into account several quantitative and qualitative oct data : retinal thickness , retinal morphology , retinal topography , macular traction , and foveal photoreceptor status . normal macular morphology and thickness not reaching the criteria for subclinical dme;early subclinical macular oedema no clinically detected retinal thickening on ophthalmoscopy , oct measured retinal thickness exceeding normal + 2sds for central fixation point and fovea;established macular oedema retinal thickening and evident morphological characteristics of oedema . no macular oedema normal macular morphology and thickness not reaching the criteria for subclinical dme ; early subclinical macular oedema no clinically detected retinal thickening on ophthalmoscopy , oct measured retinal thickness exceeding normal + 2sds for central fixation point and fovea ; established macular oedema retinal thickening and evident morphological characteristics of oedema . this includes the following : simple noncystoid macular oedema increased retinal thickness , reduced intraretinal reflectivity , irregularity of the layered structure , and flattening of the foveal depression , without presence of cystoid spaces;cystoid macular oedema the above criteria , associated with presence of well - defined intraretinal cystoid spaces : ( a)mild cystoid macular oedema cystoid spaces with horizontal diameter < 300 m , ( b)intermediate cystoid macular oedema cystoid spaces with horizontal diameter 300 m < 600 m , ( c)severe cystoid macular oedema cystoid spaces with horizontal diameter 600 m , or large confluent cavities with retinoschisis appearance;serous macular detachment any of the above , associated with serous macular detachment ( hyporeflective area under the detached neurosensory retina and over the hyperreflective line of the rpe ) . simple noncystoid macular oedema increased retinal thickness , reduced intraretinal reflectivity , irregularity of the layered structure , and flattening of the foveal depression , without presence of cystoid spaces ; cystoid macular oedema the above criteria , associated with presence of well - defined intraretinal cystoid spaces : ( a)mild cystoid macular oedema cystoid spaces with horizontal diameter < 300 m , ( b)intermediate cystoid macular oedema cystoid spaces with horizontal diameter 300 m < 600 m , ( c)severe cystoid macular oedema cystoid spaces with horizontal diameter 600 m , or large confluent cavities with retinoschisis appearance ; mild cystoid macular oedema cystoid spaces with horizontal diameter < 300 m , intermediate cystoid macular oedema cystoid spaces with horizontal diameter 300 m < 600 m , severe cystoid macular oedema cystoid spaces with horizontal diameter 600 m , or large confluent cavities with retinoschisis appearance ; serous macular detachment any of the above , associated with serous macular detachment ( hyporeflective area under the detached neurosensory retina and over the hyperreflective line of the rpe ) . this includes the following : nonsignificant macular oedema;clinically significant macular oedema , as defined by etdrs and evaluated on the oct retinal topography map . nonsignificant macular oedema ; clinically significant macular oedema , as defined by etdrs and evaluated on the oct retinal topography map . this includes the following : no macular traction presence of complete pvd ( weiss ring detected on ophthalmoscopy ) , or no pvd ( no visible posterior hyaloid line on fdoct ) , and no erm;questionable macular traction incomplete pvd with perifoveal or peripapillary adhesion and/or globally adherent erm without detectable distortion of retinal surface contour at the points of adhesion;definite macular traction incomplete pvd with perifoveal adhesion and/or focal erm with detectable distortion of retinal contour at the points of adhesion . no macular traction presence of complete pvd ( weiss ring detected on ophthalmoscopy ) , or no pvd ( no visible posterior hyaloid line on fdoct ) , and no erm ; questionable macular traction incomplete pvd with perifoveal or peripapillary adhesion and/or globally adherent erm without detectable distortion of retinal surface contour at the points of adhesion ; definite macular traction incomplete pvd with perifoveal adhesion and/or focal erm with detectable distortion of retinal contour at the points of adhesion . this includes the following : is / os and elm intact;is / os and elm with disrupted integrity . is / os and elm intact ; is / os and elm with disrupted integrity . apart from the acquisition of morphological images , oct can also detect a doppler frequency shift of reflected light , which provides information on flow and movement [ 3537 ] . wang et al . reported that reproducible and repeatable measurements of total blood flow can be obtained using doppler oct . in another study , wang et al . compared blood flow in a patient with diabetes and no retinopathy with another patient with treated proliferative retinopathy . the first subject showed a total blood flow value at the lower level of the normal range , whereas the same value in a patient with diabetic retinopathy was lower compared to healthy population . these results clearly indicate that doppler oct may play a role in noninvasive assessment of retinal blood flow in diabetic patients . the oct can also be used for visualizing the tiny blood vessels within the macula . during the american academy of ophthalmology annual meeting 2012 we presented a novel method ( oct angiography ) for the noninvasive visualization of three - dimensional retinal microcapillary network using intensity - based oct and also validated its clinical usefulness in retinal vascular diseases including diabetic retinopathy ( sikorski bl , szkulmowski m , malukiewicz g , kowalczyk a , wojtkowski m. noninvasive visualization of 3d retinal microcapillary network using oct . oct angiography proved to be capable of showing 10 micron blood vessels , revealing the vascular nonperfusion and identifying microexudates which were not otherwise visible on clinical examination and fundus photography . moreover , oct angiography can show even more capillaries in the pericentral macula than fa and allows discerning and visualizing separately the superficial and deep capillary plexus . therefore , we believe that classic structural oct examination together with oct angiography may provide a comprehensive solution in a single imaging modality in patients with diabetic maculopathy . oct can perform micrometre - resolution , cross - sectional imaging of the retina that closely approximates its histological layers . one of the huge advantages of oct is that patients find this procedure very comfortable because it is noncontact and the measurement time is very short . in patients with diabetic retinopathy oct can be successfully utilized as an objective monitoring technique of the macular thickening before and after therapy . oct is also very useful for vitreous assessment , showing whether it is attached or detached from the macula . it is helpful in detecting vitreoretinal traction that may not have been identified clinically . to summarize , oct may assist in patient selection with diabetic maculopathy who can benefit from treatment , identify what treatment is indicated , guide its implementing , and allow precise monitoring of treatment response . it seems to be the technique of choice for the early detection of macular oedema and for the followup of diabetic maculopathy .

combination anticancer therapy typically consists of drugs that target different biochemical pathways or those that act on different targets in the same pathway . here we demonstrate a new concept in combination therapy , that of enzyme activation with two compounds that hit the same biological target , but through different mechanisms . combinations of procaspase-3 activators pac-1 and 1541b show considerable synergy in activating procaspase-3 in vitro , stimulate rapid and dramatic maturation of procaspase-3 in multiple cancer cell lines , and powerfully induce caspase - dependent apoptotic death to a degree well exceeding the additive effect . in addition , the combination of pac-1 and 1541b effectively reduces tumor burden in a murine lymphoma model at dosages for which the compounds alone have minimal or no effect . these data suggest the potential of pac-1/1541b combinations for the treatment of cancer and , more broadly , demonstrate that differentially acting enzyme activators can potently synergize to give a significantly heightened biological effect . diabetic maculopathy ( dm ) is one of the major causes of vision impairment in individuals with diabetes . the traditional approach to diagnosis of dm includes fundus ophthalmoscopy and fluorescein angiography . although very useful clinically , these methods do not contribute much to the evaluation of retinal morphology and its thickness profile . that is why a new technique called optical coherence tomography ( oct ) was utilized to perform cross - sectional imaging of the retina . it facilitates measuring the macular thickening , quantification of diabetic macular oedema , and detecting vitreoretinal traction . thus , oct may assist in patient selection with dm who can benefit from treatment , identify what treatment is indicated , guide its implementing , and allow precise monitoring of treatment response . it seems to be the technique of choice for the early detection of macular oedema and for the followup of dm .

the design of the china peace - retrospective ami study has been published previously.8 in brief , we developed a nationally representative sample of hospitalizations for ami in 2001 , 2006 , and 2011 using a 2-stage random sampling design . in the first stage , we identified hospitals using a simple random sampling procedure within 5 geographical - economic strata of china : eastern - rural ; central - rural ; western - rural ; eastern - urban ; and central / western - urban regions . we used these strata because hospital volumes and clinical capacities differ between urban and rural areas as well among the 3 official geographical regions ( eastern , central , and western ) of mainland china . we combined central and western urban regions given their similar per capita income and health services capacity . in the 3 rural strata , the sampling framework consisted of the central hospital in each of the predefined rural regions ( 2010 central hospitals in 2010 rural regions ) . in the 2 urban strata , the sampling framework consisted of the highest - level hospitals in each of the predefined urban regions ( 833 hospitals in 287 urban regions ) . we randomly selected representative hospitals from 2011 to assess current practices and traced this cohort backward to 2006 and 2001 to describe temporal trends . in the second stage , using systematic random sampling procedures , we drew cases from each sampled hospital using the local hospital database for patients with ami in 2001 , 2006 , and 2011 . ami cases were identified using a principal discharge diagnosis of ami based on international classification of diseases versions 9 or 10 , given that hospitals in china are mandated by the ministry of health to list this information on the first page of the medical record , and in rare cases when such information was not available , we confirmed the diagnosis through medical record review . data abstraction quality was monitored by randomly auditing 5% of the medical charts , with overall accuracy exceeding 98% . the central ethics committee at the china national center for cardiovascular diseases , or local internal ethics committees approved the china peace - retrospective ami study . the funder of the study had no role in study design , data collection , data analysis , data interpretation , or writing of the report . we limited study samples to patients potentially eligible for spironolactone , namely , those with known hf or diabetes at discharge . patients who had a length of hospital stay shorter than 24 hours were excluded to ensure that all patients had sufficient opportunity to receive spironolactone . subsequently , we classified patients into 1 of 4 groups : the ideal group consisted of patients with a documented lvef 40% and no contraindication to spironolactone ; the contraindicated group consisted of patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; the not indicated group consisted of patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; and the unknown indications group consisted of patients whose lvef was not measured during the hospitalization . data elements collected included demographic information , medical history , patient characteristics at presentation , hospital characteristics , laboratory parameters , concomitant therapy , and documented diagnosis . cardiovascular risk ( cvr ) factors were coded as present if diagnosed before or during admission ; clinical characteristics , including vital signs , represented those recorded on admission . to capture the laboratory values likely to influence the decision about spironolactone therapy in patients who received the drug , we used the last potassium and creatinine values before administration of medication . for patients who ultimately did not receive spironolactone , we used the highest lab value recorded during hospitalization to ensure identification of any possible contraindication . lvef values were based on echocardiography , radionuclide angiography , or computerized tomography coronary angiography . to examine temporal trends in spironolactone therapy , we used the cochran - armitage tests and applied weights proportional to the inverse sampling fraction of hospitals to account for differences in the sampling fraction for each time period . when exploring patient and hospital characteristics associated with the use of spironolactone , categorical variables were expressed as frequencies and percentages and analyzed using chi - square tests . among all the variables , missing data were rare and occurred only for the age variable ( 0.1% ) , which was imputed to the overall median to avoid case - wise deletion . we used logistic regression models to identify predictors independently associated with spironolactone use in different patient groups by indications . variables in models include demographic characteristics , cvr factors , medical history , conditions and vital signs on admission , estimated glomerular filtration rate ( egfr ) , ami type , economic - geographical region , rural / urban region , and years . in addition , we also included hospital characteristics , such as teaching status and percutaneous coronary intervention ( pci ) capability ( table 1 ) . a generalized estimating equation model was developed to account for clustering of patients within hospitals . all variables in the bivariate model were included in the multivariable model except those with frequencies under 1% . bivariate analysis of characteristics associated with spironolactone therapy among ideal patients ace inhibitor indicates angiotensin - converting enzyme inhibitor ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; egfr , estimated glomerular filtration rate ; nstemi , non - st - segment elevation myocardial infarction ; pci , percutaneous coronary intervention ; sbp , systolic blood pressure ; stemi , st - segment elevation myocardial infarction . all comparisons were 2-tailed , with p<0.05 considered statistically significant . all statistical analyses were performed using sas software ( version 9.2 ; sas institute , cary , nc ) and r software ( version 3.0.2 ; r foundation for statistical computing , vienna , austria ) . the design of the china peace - retrospective ami study has been published previously.8 in brief , we developed a nationally representative sample of hospitalizations for ami in 2001 , 2006 , and 2011 using a 2-stage random sampling design . in the first stage , we identified hospitals using a simple random sampling procedure within 5 geographical - economic strata of china : eastern - rural ; central - rural ; western - rural ; eastern - urban ; and central / western - urban regions . we used these strata because hospital volumes and clinical capacities differ between urban and rural areas as well among the 3 official geographical regions ( eastern , central , and western ) of mainland china . we combined central and western urban regions given their similar per capita income and health services capacity . in the 3 rural strata , the sampling framework consisted of the central hospital in each of the predefined rural regions ( 2010 central hospitals in 2010 rural regions ) . in the 2 urban strata , the sampling framework consisted of the highest - level hospitals in each of the predefined urban regions ( 833 hospitals in 287 urban regions ) . we randomly selected representative hospitals from 2011 to assess current practices and traced this cohort backward to 2006 and 2001 to describe temporal trends . in the second stage , using systematic random sampling procedures , we drew cases from each sampled hospital using the local hospital database for patients with ami in 2001 , 2006 , and 2011 . ami cases were identified using a principal discharge diagnosis of ami based on international classification of diseases versions 9 or 10 , given that hospitals in china are mandated by the ministry of health to list this information on the first page of the medical record , and in rare cases when such information was not available , we confirmed the diagnosis through medical record review . data abstraction quality was monitored by randomly auditing 5% of the medical charts , with overall accuracy exceeding 98% . the central ethics committee at the china national center for cardiovascular diseases , or local internal ethics committees approved the china peace - retrospective ami study . the funder of the study had no role in study design , data collection , data analysis , data interpretation , or writing of the report . we limited study samples to patients potentially eligible for spironolactone , namely , those with known hf or diabetes at discharge . patients who had a length of hospital stay shorter than 24 hours were excluded to ensure that all patients had sufficient opportunity to receive spironolactone . subsequently , we classified patients into 1 of 4 groups : the ideal group consisted of patients with a documented lvef 40% and no contraindication to spironolactone ; the contraindicated group consisted of patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; the not indicated group consisted of patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; and the unknown indications group consisted of patients whose lvef was not measured during the hospitalization . data elements collected included demographic information , medical history , patient characteristics at presentation , hospital characteristics , laboratory parameters , concomitant therapy , and documented diagnosis . cardiovascular risk ( cvr ) factors were coded as present if diagnosed before or during admission ; clinical characteristics , including vital signs , represented those recorded on admission . to capture the laboratory values likely to influence the decision about spironolactone therapy in patients who received the drug , we used the last potassium and creatinine values before administration of medication . for patients who ultimately did not receive spironolactone , we used the highest lab value recorded during hospitalization to ensure identification of any possible contraindication . lvef values were based on echocardiography , radionuclide angiography , or computerized tomography coronary angiography . to examine temporal trends in spironolactone therapy , we used the cochran - armitage tests and applied weights proportional to the inverse sampling fraction of hospitals to account for differences in the sampling fraction for each time period . when exploring patient and hospital characteristics associated with the use of spironolactone , categorical variables were expressed as frequencies and percentages and analyzed using chi - square tests . among all the variables , missing data were rare and occurred only for the age variable ( 0.1% ) , which was imputed to the overall median to avoid case - wise deletion . we used logistic regression models to identify predictors independently associated with spironolactone use in different patient groups by indications . variables in models include demographic characteristics , cvr factors , medical history , conditions and vital signs on admission , estimated glomerular filtration rate ( egfr ) , ami type , economic - geographical region , rural / urban region , and years . in addition , we also included hospital characteristics , such as teaching status and percutaneous coronary intervention ( pci ) capability ( table 1 ) . a generalized estimating equation model was developed to account for clustering of patients within hospitals . all variables in the bivariate model were included in the multivariable model except those with frequencies under 1% . bivariate analysis of characteristics associated with spironolactone therapy among ideal patients ace inhibitor indicates angiotensin - converting enzyme inhibitor ; ami , acute myocardial infarction ; arb , angiotensin receptor blocker ; egfr , estimated glomerular filtration rate ; nstemi , non - st - segment elevation myocardial infarction ; pci , percutaneous coronary intervention ; sbp , systolic blood pressure ; stemi , st - segment elevation myocardial infarction . all statistical analyses were performed using sas software ( version 9.2 ; sas institute , cary , nc ) and r software ( version 3.0.2 ; r foundation for statistical computing , vienna , austria ) . the nationally representative samples described in the china peace - retrospective ami study consisted of 16 100 patients hospitalized for ami in 162 hospitals across china ( figure 1a ) , with the 2011 sample representing 245 720 patients across china . after excluding patients with a length of stay shorter than 24 hours , and those without hf or diabetes when discharged , we identified 6906 patients ( 12.2% in 2001 , 27.7% in 2006 , and 60.1% in 2011 ) who were potentially eligible for spironolactone ( figure 1b ) . across all years , median age was 69 years ( interquartile range , 59 to 76 ) and 35.8% were female . among these patients , 44.2% had diabetes , and almost three quarters of patients ( 73.9% ) had hf - among them cvr factors were common : 57.9% had hypertension ( htn ) , 29.8% were current smokers , and 28.0% had coronary artery disease . a , flow diagram showing the process used to produce a nationally representative sampling of hospitals in china . b , flow diagram showing the approach to classify patients into 4 groups according to their indications for spironolactone . n represents number of patients . there were notable changes in the relative proportion of the 4 patient groups over time ( figure 2 ) . for example , the proportion of ideal patients doubled from 2001 to 2006 ( 4.5% to 9.1% ) and remained stable thereafter ( 10.2% in 2011 ) . in contrast , the proportion of contraindicated patients varied little across the 3 years ( 13.0% , 11.9% , and 10.2% in 2001 , 2006 , and 2011 , respectively ) . patients increased markedly over the years ( from 15.6% in 2001 to 46.8% in 2011 ; p<0.001 for trend ) , whereas that of unknown indications acute myocardial infarction patients with heart failure or diabetes grouped by their eligibility for spironolactone in 2001 , 2006 , and 2011 . ideal : patients with a left ventricular ejection fraction ( lvef ) 40% and without contraindications to spironolactone ; contraindicated : patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; not indicated : patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; unknown indications : patients whose lvef was not measured during the hospitalization . overall , the weighted rate of spironolactone use in 2011 differed among patients in each group : 72.4% in ideal ; 27.5% in contraindicated ; 38.3% in not indicated ; and 35.1% in unknown indications . spironolactone use increased in all groups over the past decade : among ideal patients , the weighted rate of use increased from 28.6% in 2001 to 68.5% in 2006 and to 72.4% in 2011 ( p<0.001 for trend ) , whereas for contraindicated patients it increased from 11.4% in 2001 to 22.4% in 2006 and to 27.5% in 2011 ( p=0.002 for trend ) . similar increases were observed among not indicated patients ( p=0.007 for trend ) and unknown indications patients ( p<0.001 for trend ; figure 3 ) . given that spironolactone can also be used to treat htn or as a concomitant therapy in hf with reduced lvef , we performed a post - hoc analysis describing spironolactone use in a specific subgroup of not indicated patients , namely , those with neither htn nor hf . in this subgroup of patients in 2011 , 17.2% received spironolactone . spironolactone use ( weighted ) among different groups of acute myocardial infarction patients with heart failure or diabetes according to their eligibility for spironolactone in 2001 , 2006 , and 2011 . ideal : patients with a left ventricular ejection fraction ( lvef ) 40% and without contraindications to spironolactone ; contraindicated : patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; not indicated : patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; unknown indications : patients whose lvef was not measured during the hospitalization . bivariate analysis of the factors associated with the use of spironolactone among ideal patients is shown in table 1 . all characteristics were entered into the multivariable model to determine independent predictors of use ( figure 4 ) . specifically , older patients ( 65 year ) were more likely to be treated than younger patients ( 72.2% vs. 55.1% ; or , 2.07 ; 95% ci , 1.30 to 3.30 ) . patients with htn or with symptoms of hf at admission were both more likely to receive spironolactone ( 70.2% vs. 61.1% ; or , 1.57 ; 95% ci , 1.11 to 2.23 and 72.9% vs. 52.1% ; or , 2.00 ; 95% ci , 1.18 to 3.36 , respectively ) than those without these comorbidities . patients in pci - capable hospitals were more likely to be treated ( 71.1% vs. 51.6% ; or , 2.12 ; 95% ci , 1.12 to 4.02 ) . . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . egfr indicates estimated glomerular filtration rate ; or , odds ratio ; pci , percutaneous coronary intervention . characteristics in multivariable analysis that remained independently associated with the use of spironolactone in the other 3 groups are shown in figures5 through 7 . contraindicated and unknown indications groups ( or , 1.84 ; 95% ci , 1.15 to 2.94 and or , 1.74 ; 95% ci , 1.31 to 2.29 , respectively ) . patients > 65 years old and patients with htn had a high likelihood of receiving spironolactone among both not indicated and unknown indication groups . across all groups , patients with symptoms of hf at admission to the hospital were more likely to be treated with spironolactone ( contraindicated : or , 3.26 ; 95% ci , 2.05 to 5.16 ; unknown indications : or , 2.60 ; 95% ci , 2.12 to 3.18 ; not indicated : or , 2.16 ; 95% ci , 1.73 to 2.69 ) . factors associated with spironolactone therapy among contraindicated patients in multivariable model . . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . factors associated with spironolactone therapy among not indicated patients in the multivariable model . . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . factors associated with spironolactone therapy among unknown indications patients in the multivariable model . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . egfr indicates estimated glomerular filtration rate ; or , odds ratio ; sbp , systolic blood pressure . the nationally representative samples described in the china peace - retrospective ami study consisted of 16 100 patients hospitalized for ami in 162 hospitals across china ( figure 1a ) , with the 2011 sample representing 245 720 patients across china . after excluding patients with a length of stay shorter than 24 hours , and those without hf or diabetes when discharged , we identified 6906 patients ( 12.2% in 2001 , 27.7% in 2006 , and 60.1% in 2011 ) who were potentially eligible for spironolactone ( figure 1b ) . across all years , median age was 69 years ( interquartile range , 59 to 76 ) and 35.8% were female . among these patients , 44.2% had diabetes , and almost three quarters of patients ( 73.9% ) had hf - among them cvr factors were common : 57.9% had hypertension ( htn ) , 29.8% were current smokers , and 28.0% had coronary artery disease . a , flow diagram showing the process used to produce a nationally representative sampling of hospitals in china . b , flow diagram showing the approach to classify patients into 4 groups according to their indications for spironolactone . n represents number of patients . there were notable changes in the relative proportion of the 4 patient groups over time ( figure 2 ) . for example , the proportion of ideal patients doubled from 2001 to 2006 ( 4.5% to 9.1% ) and remained stable thereafter ( 10.2% in 2011 ) . in contrast , the proportion of contraindicated patients varied little across the 3 years ( 13.0% , 11.9% , and 10.2% in 2001 , 2006 , and 2011 , respectively ) . patients increased markedly over the years ( from 15.6% in 2001 to 46.8% in 2011 ; p<0.001 for trend ) , whereas that of unknown indications acute myocardial infarction patients with heart failure or diabetes grouped by their eligibility for spironolactone in 2001 , 2006 , and 2011 . ideal : patients with a left ventricular ejection fraction ( lvef ) 40% and without contraindications to spironolactone ; contraindicated : patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; not indicated : patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; unknown indications : patients whose lvef was not measured during the hospitalization . overall , the weighted rate of spironolactone use in 2011 differed among patients in each group : 72.4% in ideal ; 27.5% in contraindicated ; 38.3% in not indicated ; and 35.1% in unknown indications . spironolactone use increased in all groups over the past decade : among ideal patients , the weighted rate of use increased from 28.6% in 2001 to 68.5% in 2006 and to 72.4% in 2011 ( p<0.001 for trend ) , whereas for contraindicated patients it increased from 11.4% in 2001 to 22.4% in 2006 and to 27.5% in 2011 ( p=0.002 for trend ) . similar increases were observed among not indicated patients ( p=0.007 for trend ) and unknown indications patients ( p<0.001 for trend ; figure 3 ) . given that spironolactone can also be used to treat htn or as a concomitant therapy in hf with reduced lvef , we performed a post - hoc analysis describing spironolactone use in a specific subgroup of not indicated patients , namely , those with neither htn nor hf . in this subgroup of patients in 2011 , 17.2% received spironolactone . spironolactone use ( weighted ) among different groups of acute myocardial infarction patients with heart failure or diabetes according to their eligibility for spironolactone in 2001 , 2006 , and 2011 . ideal : patients with a left ventricular ejection fraction ( lvef ) 40% and without contraindications to spironolactone ; contraindicated : patients with a contraindication ( serum potassium > 5 mmol / l , or serum creatinine > 2.5 mg / dl [ men ] or > 2.0 mg / dl [ women ] , or documented allergy to spironolactone ) ; not indicated : patients with neither indication ( ie , lvef > 40% ) nor contraindication to spironolactone ; unknown indications : patients whose lvef was not measured during the hospitalization . bivariate analysis of the factors associated with the use of spironolactone among ideal patients is shown in table 1 . all characteristics were entered into the multivariable model to determine independent predictors of use ( figure 4 ) . specifically , older patients ( 65 year ) were more likely to be treated than younger patients ( 72.2% vs. 55.1% ; or , 2.07 ; 95% ci , 1.30 to 3.30 ) . patients with htn or with symptoms of hf at admission were both more likely to receive spironolactone ( 70.2% vs. 61.1% ; or , 1.57 ; 95% ci , 1.11 to 2.23 and 72.9% vs. 52.1% ; or , 2.00 ; 95% ci , 1.18 to 3.36 , respectively ) than those without these comorbidities . patients in pci - capable hospitals were more likely to be treated ( 71.1% vs. 51.6% ; or , 2.12 ; 95% ci , 1.12 to 4.02 ) . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . egfr indicates estimated glomerular filtration rate ; or , odds ratio ; pci , percutaneous coronary intervention . characteristics in multivariable analysis that remained independently associated with the use of spironolactone in the other 3 groups are shown in figures5 through 7 . contraindicated and unknown indications groups ( or , 1.84 ; 95% ci , 1.15 to 2.94 and or , 1.74 ; 95% ci , 1.31 to 2.29 , respectively ) . patients > 65 years old and patients with htn had a high likelihood of receiving spironolactone among both not indicated and unknown indication groups . across all groups , patients with symptoms of hf at admission to the hospital were more likely to be treated with spironolactone ( contraindicated : or , 3.26 ; 95% ci , 2.05 to 5.16 ; unknown indications : or , 2.60 ; 95% ci , 2.12 to 3.18 ; not indicated : or , 2.16 ; 95% ci , 1.73 to 2.69 ) . . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . factors associated with spironolactone therapy among not indicated patients in the multivariable model . . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . factors associated with spironolactone therapy among unknown indications patients in the multivariable model . the adjusted odds ratio of 1 shows no difference to receive spironolactone therapy among ideal patients . each dot represents the point estimate of the effect of that variable in the model ; the line shows the 95% confidence interval ( ci ) . egfr indicates estimated glomerular filtration rate ; or , odds ratio ; sbp , systolic blood pressure . in this national quality assessment analyzing spironolactone use among patients with ami in china , we found that spironolactone use increased over time . however , suboptimal patient identification and selection were detected throughout the study period and persisted in 2011 . although more patients underwent lvef assessment , which is necessary to determine their eligibility for spironolactone , one third of patients did not have an lvef assessment during their hospitalization for ami in 2011 . spironolactone use among patients who may not benefit and those with contraindications was common and such use increased significantly over time . our findings indicate that the chinese health care system rapidly responded to new information that highlighted the utility of spironolactone , but also appears to have driven increased use among patients who lack a strong indication , albeit at a lower rate than in ideal patients . these findings highlight an opportunity for hospitals in china to improve the translation of evidence into clinical practice . to our knowledge , this is the first comprehensive , nationally representative quality assessment of spironolactone use in ami in china . previous studies evaluating the use of aldosterone antagonists among ami patients in other countries have focused only on patients with definite indications for treatment.911 in contrast , our study describes to what extent patients with ami are evaluated for spironolactone and shows that spironolactone is used not only among patients with indications , but also those with contraindications , without indications , and with indications unknown . additionally , the use of a nationally representative sample ensures that the findings of this analysis are broadly applicable across china and can serve as the basis for future quality improvement initiatives . lvef assessment among patients with ami improved over the past decade , which created more opportunities to consider spironolactone therapy ; however , further improvement is possible . in 2001 , indications for two thirds of patients were unknown and only 4.5% of the cohort was classified as ideal patients increased to 10% ; however , one third of patients still lacked an lvef assessment . in comparison , among patients in the united states with ami from 2007 to 2009 , the rate of lvef assessment was reported to be 91.0%.12 it should be noted that lvef assessment is a critical component of the care of patients with ami because it enables risk stratification and guides the prescription of other therapies as well , for instance , inhibitors of angiotensin converting enzyme ( ace ) . the increasing use of spironolactone among ideal patients in china over time is encouraging . in 2001 , spironolactone use among ideal patients was no better than other groups , but it increased sharply thereafter , possibly in response to the ephesus study , which was published after 2001 and clearly supported the use of aldosterone antagonists in this patient population.6 there are no previous studies in china with which to compare our results . however , a registry - based study in spain reported that 54.8% patients with ami and hf received aldosterone antagonist in hospital between 2006 and 20089 ; and in the united states , the prescription rate of aldosterone antagonists at discharge among patients with ami and reduced lvef was only approximately 15% from 2009 to 2010.10,11 although the utilization seems to be better in china than in other countries , there remains room for further improvement , given the potential benefit of this agent . the substantial use of spironolactone among patients with contraindications nearly 1 in 4 patients was concerning and indicates a gap in the patient selection process that can expose patients to potential harm , such as worsening hyperkalemia or significant renal dysfunction.4,13 it must be noted that there may be circumstances in which spironolactone use is contraindicated according to the guidelines , yet clinicians perceive that the benefit of the drug will outweigh its risks . for example , some recently published studies demonstrate that the benefit of aldosterone antagonists may offset its risk in the setting of moderate hyperkalemia.14,15 nonetheless , the rising proportion of contraindicated patients being treated with spironolactone is consistent with a study in the united states and suggests that the growth in use may , at times , be indiscriminate.16 spironolactone was frequently used among patients without clear indications , namely , those with lvef > 40% . it should be noted that the recent treatment of preserved cardiac function heart failure with an aldosterone antagonist study found no benefit of spironolactone use in patients with hf and a preserved lvef.17 in the subgroup of patients without any identifiable indication , including htn or systolic hf , 17.2% were treated , suggesting that a substantial number of patients are receiving a drug that has limited benefit for their condition , and with a potential for adverse effects . in all 4 groups , such a pattern indicates that the presence of hf was a common and important trigger for spironolactone prescription by clinicians and implies a wide acceptance of the drug in hf . additionally , the steady rate of loop diuretic use among patients with hf indicates that spironolactone was not being systematically substituted for loop diuretics , and that other factors are driving spironolactone uptake . the selection issues identified in this analysis may impede the transfer of benefit to appropriate patients . quality improvement initiatives emphasizing the appropriate use of spironolactone are warranted to improve patient selection and avoid adverse events . however , to ensure accurate abstraction , we had strict definitions for all variables and employed quality control procedures to insure that the abstraction accuracy reached 98% . second , we may not have captured all patients with contraindications owing to inadequate physician documentation or unmeasured serum chemistry , which may lead to errors in the estimation of ideal patients and contraindicated patients . third , the benefit of aldosterone antagonists for ami patients was demonstrated in the ephesus study , which studied eplerenone , rather than spironolactone , which is the only aldosterone antagonist available in china . however , the structural similarity of spironolactone and eplerenone suggests that they may have similar efficacy , and guidelines endorse the use of both drugs for patients with ami . last , because data were abstracted from deidentified medical charts , we were unable to determine whether some patients were included in multiple study years , or had multiple admissions during the same year ; however , given the 5-year difference between the 3 time points , and the random sampling method , the number of such patients is likely to be minimal . first , our analysis used data abstracted from medical records . however , to ensure accurate abstraction , we had strict definitions for all variables and employed quality control procedures to insure that the abstraction accuracy reached 98% . second , we may not have captured all patients with contraindications owing to inadequate physician documentation or unmeasured serum chemistry , which may lead to errors in the estimation of ideal patients and contraindicated patients . third , the benefit of aldosterone antagonists for ami patients was demonstrated in the ephesus study , which studied eplerenone , rather than spironolactone , which is the only aldosterone antagonist available in china . however , the structural similarity of spironolactone and eplerenone suggests that they may have similar efficacy , and guidelines endorse the use of both drugs for patients with ami . last , because data were abstracted from deidentified medical charts , we were unable to determine whether some patients were included in multiple study years , or had multiple admissions during the same year ; however , given the 5-year difference between the 3 time points , and the random sampling method , the number of such patients is likely to be minimal . we identified opportunities to optimize the use of spironolactone post - ami in chinese clinical practice , including wider lvef assessment , more - careful selection of patients , and increasing the utilization among ideal patients . our findings shed light on existing practice patterns in the treatment of ami in china , serve as the basis for future quality assessment efforts , and illuminate the barriers to more - appropriate use of evidence - based therapies for all countries seeking opportunity to optimize care . this project was partly supported by the research special fund for public welfare industry of health ( 201202025 ) from the national health and family planning commission of china . dr krumholz is supported by grant u01hl105270 - 05 ( center for cardiovascular outcomes research at yale university ) from the national heart , lung , and blood institute . dr ross is supported by the national institute on aging ( k08ag032886 ) and by the american federation for aging research through the paul b. beeson career development award program . the funders had no role in study design , data collection and analysis , decision to publish , or preparation of the manuscript . dr krumholz reports being the recipient of research grants from medtronic and johnson & johnson , through yale university , to develop methods of clinical trial data sharing , and the chair of a cardiac scientific advisory board for unitedhealth . dr ross reports that he is a member of a scientific advisory board for fair health , inc . dr masoudi receives salary support from the american college of cardiology for his role as the senior medical officer of the national cardiovascular data registries . appendix s1 . members of the china peace collaborative group - retrospective ami study site investigators and china peace study consultants . to our knowledge the most recent article on celiac dissection was published in 2015 and reported 24 known cases of spontaneous isolated celiac trunk dissection . in our literature review we did not identify a single case of isolated celiac artery dissection that presented during a hypertensive emergency . we present a case of hypertensive emergency induced spontaneous isolated celiac trunk dissection , which was managed nonoperatively with a labetalol drip . the definition of an arterial dissection is the cleavage of the arterial wall by an intramural hematoma located between two elastic layers , . as we are reporting the 25th known case of celiac artery dissection many of the other reported cases involve other arteries such as the common hepatic , splenic , superior mesenteric , and gastroduodenal , , , , , , . additionally , while some of those cases reported hypertension as a risk factor , no other case presents as hypertensive emergency with an isolated celiac artery dissection . the first case of visceral artery dissection involved the superior mesenteric artery ( sma ) and was reported in 1947 , while the first celiac artery dissection was not reported until 1959 . when they occur in the visceral arteries , the most common location is within the sma , . celiac artery dissection has been described in blunt abdominal trauma , but it only accounts for 12 percent of all visceral vascular lesions . to our knowledge the most recent article on celiac dissection was published in 2015 and reported 24 known cases of spontaneous isolated celiac trunk dissection . in our literature review we did not identify a single case of isolated celiac artery dissection that presented during a hypertensive emergency . we present a case of hypertensive emergency induced spontaneous isolated celiac trunk dissection , which was managed nonoperatively with a labetalol drip . the definition of an arterial dissection is the cleavage of the arterial wall by an intramural hematoma located between two elastic layers , . as we are reporting the 25th known case of celiac artery dissection many of the other reported cases involve other arteries such as the common hepatic , splenic , superior mesenteric , and gastroduodenal , , , , , , . additionally , while some of those cases reported hypertension as a risk factor , no other case presents as hypertensive emergency with an isolated celiac artery dissection . the first case of visceral artery dissection involved the superior mesenteric artery ( sma ) and was reported in 1947 , while the first celiac artery dissection was not reported until 1959 . when they occur in the visceral arteries , the most common location is within the sma , . celiac artery dissection has been described in blunt abdominal trauma , but it only accounts for 12 percent of all visceral vascular lesions . a 43 year - old man with a past medical history of uncontrolled hypertension , for which he had reportedly been non - compliant with follow - up , presented with complaints of severe , sudden - onset epigastric pain which was non - radiating and constant for 1 h prior to arrival . he reported moving his bowels prior to the onset of symptoms , and noted passing flatus after the onset of symptoms . the patient denied a history of recent abdominal trauma . on initial examination his vital signs consisted of a temperature of 98.1 fahrenheit , heart rate of 78 , respiratory rate of 16 , blood pressure of 234/144 , and 99% oxygen saturation on room air . his hypertensive emergency in the setting of severe abdominal pain necessitated an emergent vascular surgery consultation . examination of the abdomen revealed a soft , non - distended , and obese abdomen . the patient was diffusely tender to palpation , with maximum tenderness overlying the epigastric region . there was no evidence of voluntary guarding or rebound , and he did not display peritoneal symptoms . pulse examination was positive for 2 + radial , femoral , dorsalis pedis , and posterior tibial pulses bilaterally . there was a well - healed , vertical , surgical scar in the left groin from an unspecified procedure secondary to a remote history of traumatic stab wound . laboratory analyses ( complete blood count , coagulation parameters , comprehensive metabolic panel , lactic acid , and troponin i ) were all within normal limits . contrast enhanced ( oral and intravenous ) computed tomography of the chest and abdomen / pelvis demonstrated normal course and caliber of the great vessels , ascending aorta , descending aorta , and abdominal aorta without aneurysmal dilatation . there was no evidence of displaced intimal calcification , intramural hematoma , or dissection flap to suggest an aortic dissection . an intimal flap was noted within the celiac trunk , starting at the origin and extending into the left gastric , splenic , and the common hepatic arteries . the true and false lumens of the celiac trunk appeared well perfused . from the level of the bifurcation of the celiac trunk , perfusion of the true lumen of the left gastric , splenic , and common hepatic artery was noted . the superior mesenteric artery , bilateral renal arteries , inferior mesenteric artery , common iliac arteries , and internal / external iliac arteries were patent without aneurysmal dilatation . there were no signs of ischemic change of the liver and stomach , and the pancreas was of normal size and contour . the patient was treated with 8 mg of morphine with only slight relief from his symptoms . the patient was also treated with 10 mg labetalol iv and his systolic blood pressure improved into the 140s . an additional 6 mg of morphine was administered for persistent abdominal pain and an additional 10 mg labetalol was prescribed . the patient was admitted to the cardiac intensive care unit for aggressive blood pressure control and was initiated on a labetalol drip to maintain systolic blood pressure less than 120 mm hg . with aggressive blood pressure control throughout the evening of hospital day 0 and hospital day 1 , the patient s abdominal pain decreased in intensity . he was then started on a regular diet , which he consumed without nausea , vomiting , or exacerbation of his pain . on hospital day 1 , the patient was weaned off of the labetalol drip , and his blood pressure medication regimen was transitioned to a by mouth route of hydralazine , labetalol , and amlodipine . the most common symptom in patients with celiac artery dissection is acute or chronic epigastric or abdominal pain , , , . in many of the case studies the patient presented to the emergency department with sudden onset epigastric pain , , . other manifestations include obstructive jaundice , pancreatitis , intestinal angina , tachycardia , hemorrhage , and hypertension , , . interestingly , patients do not usually present with nausea , vomiting , or peritoneal symptoms , , . another case study had a patient present with hematemesis , melena , and postprandial pain . one of the more unique presentations was in a patient who had a history of long - term energy drink consumption and intense exercise routines . patients with celiac artery dissection may also have a self - limited course of these aforementioned symptoms . extensive of the dissection may occur into adjacent arterial walls , including those of the splenic artery and proximal hepatic arteries . renal arteries and the splenic artery may also infarct in the first week after celiac dissection . the hepatic artery may develop an aneurysm . there was one patient who experienced a sequential sma dissection in the subsequent week during his hospital stay our patient did not experience any of these complications .. the crux of the diagnosis of this condition relies on contrast enhanced ct ( ct ) . other modalities that can be used are ct angiography ( cta ) , magnetic resonance imaging ( mri ) , magnetic resonance angiography ( mra ) , and doppler ultrasonography ( us ) . cta is used for definitive diagnosis as it allows precise determination of collateral circulation , . us is of some efficacy because one can assess areas of abnormal flow in the proper habitus . other cases have found infiltration of the fat surrounding the celiac artery or celiac artery aneurysm , . intramural hematoma has been identified on ct and may lead to moderate narrowing of the vessel . another reported a length of 812 millimeters . in the one patient that presented with upper gastrointestinal hemorrhage ct showed enhancement of perigastric and gastric intramural vascular collaterals due to chronic ischemia secondary to celiac artery dissection . another case study repeated the ct scan as early as 12 h later . regardless of the timeline chosen , it is imperative to have serial ct scans to monitor for the potential serious complications of celiac artery dissection . there are many identifiable and accepted risk factors for celiac artery dissection , , , , , , . hypertension is an identified risk factor , though celiac artery dissection as the presenting symptom for hypertensive emergency has yet to be described until now . in addition to hypertension , the other known risk factors are atherosclerotic disease , abdominal aortic aneurysm , cystic medial necrosis , fibromuscular dysplasia , connective tissue disorders such as ehlers - danlos syndrome , trauma , previous abdominal surgery , stenosis or occlusion of a major artery , syphilis , polyarteritis nodosa , and pregnancy , , , , . one case series noted that the two most common risk factors for celiac artery dissection were hypertension followed by vasculitis ( particularly polyarteritis nodosa ) . anti - inflammatory drugs and steroids may be indicated to treated underlying conditions such as polyarteritis nodosa . the duration of anticoagulant and antiplatelet therapy is variable but it is thought 36 months is sufficient . , for anticoagulation heparin or lovenox bridging to warfarin is recommended , . anticoagulation is essential in order to prevent thromboembolic complications , . in order to reduce the risk of propagation of the dissection and rupture , one retrospective review of 19 patients with celiac artery dissection , published in 2015 , notes that long term anticoagulation is not needed in patients undergoing endovascular stenting . complications such as occlusive lesions , aneurysm formation , arterial rupture , hemodynamic instability , persistent pain , uncontrolled hypertension , ischemia , end organ damage , or dissection extensive are indications for operative treatment , , , . when surgical intervention is necessary , a vascular surgeon should be consulted emergently . operative treatment may entail resection and anastomosis , prosthetic bypass , or endovascular stenting , , . follow - up for the patient population with celiac artery dissection should include ct scan with iv contrast at 3- and 6-month intervals to evaluate for stability of the dissection . earlier imaging may be obtained should patients become increasingly symptomatic on a more accelerated timeline . in patients with contraindications to iv contrast injection , ultrasound in conjunction with mri may suffice as an alternative option . it is reported that celiac artery dissection has a poor prognosis . this prognosis , though , depends on the extent of involvement of subsegmental branches , . involvement of the renal , hepatic , or splenic arteries can cause detrimental end - organ damage . going forward his prognosis will ultimately depend on control of his blood pressure . in the event that his symptoms recur , he will be a surgical candidate ( fig . 1 , fig . 2 , fig . 3 , fig . to the best of our knowledge , we present the 25th case of isolated celiac artery dissection . this is the first case of hypertensive emergency induced spontaneous isolated celiac trunk dissection in literature . visceral artery dissection is a rare commodity , and celiac artery dissection is less common than dissection in the sma . it is a difficult diagnosis to make on history and physical alone , necessitating contrast enhanced ct imaging . in patients who present with acute onset abdominal pain in the setting of hypertension , we recommend an immediate contrast enhanced ct scan . in patients with contraindications no research conducted on patient and he has given approval - steven d. kozusko on behalf of all authors . written informed consent was obtained from the patient for publication of this case report and accompanying images - steven d. kozusko on behalf of all authors . swergold data collection dr . swergold data analysis or interpretation dr . swergold writing the paper dr . kozusko oversight dr . sturt steven d. kozusko on behalf of all authors .

backgroundspironolactone , the only aldosterone antagonist available in china , improves outcomes in acute myocardial infarction ( ami ) among patients with systolic dysfunction and either diabetes or heart failure ( hf ) . however , national practice patterns in the use of spironolactone in china are unknown.methods and resultsfrom a nationally representative sample of ami patients from in 2001 , 2006 , and 2011 , we identified 6906 patients with either diabetes or hf and classified them into 1 of 4 groups according to their eligibility for spironolactoneideal(left ventricular ejection fraction [ lvef ] 40% and without contraindications ) , contraindicated , not indicated ( neither ideal nor contraindicated ) , and unknown indications ( lvef unmeasured)to determine how frequently patient eligibility for this drug is assessed in the hospital , how it is used in several groups , and to identify factors associated with the use in these groups . from 2001 to 2011 , the proportion of patients whose eligibility for spironolactone was not assessed decreased ( 66.9% in 2001 to 32.8% in 2011 ) . spironolactone use significantly increased among ideal patients over this period ( 28.6% to 72.4% ; p<0.001 for trend ) , but also in contraindicated patients ( 11.4% to 27.5% ; p=0.002 for trend ) and in other patients groups ( not indicated : 27.5% to 38.3% ; unknown indications : 21.3% to 35.1% ; both p<0.01 for trend ) . in all 4 groups , patients presenting with hf on admission were more likely to receive spironolactone.conclusionsalthough the appropriate use of spironolactone and assessment of eligibility increased in china over the past decade , there remains marked opportunities for improvement.clinical trial registrationurl : http://www.clinicaltrials.gov unique identifier : nct01624883 . highlightsto the best of our knowledge , we present the 25th case of isolated celiac artery dissection.this is the first case of hypertensive emergency induced spontaneous isolated celiac trunk dissection in literature.visceral artery dissection is a rare commodity , and celiac artery dissection is less common than dissection in the sma.it is a difficult diagnosis to make on history and physical alone , necessitating contrast enhanced ct imaging.our patient was managed nonoperatively with a labetalol drip and did well .

in 2008 , a 30-year - old primigravida physician experienced intrauterine fetal demise ( iufd ) at 20 weeks gestation , as established by last menstrual period and confirmed by ultrasound at 8 weeks . the patient spent most of her pregnancy providing direct patient care . in october 2007 , during gestational weeks 26 , she was exposed to at least 2 patients with presumed influenza . she reported symptoms of fever , myalgia , headache , and cough during gestational week 4 . she had no testing for influenza , did not receive antiviral therapy , and took acetaminophen ; her symptoms resolved . an ultrasound at 18 weeks demonstrated early growth restriction and oligohydramnios with normal appearing fetal kidneys and head . the patient had an unremarkable medical history , did not smoke , and was normotensive throughout pregnancy . microscopically , histiocytes were abundant in the maternal space ( chronic intervillositis ) and were noted within the fetal chorionic villi ( hofbauer cells ; figure 1 , panel a ) . histologic analysis of fetal autopsy specimens showed scattered luminal histiocytes of the lung and gut . no other inflammatory changes or viral inclusions were identified in the placenta or fetal organs . tissue sample from 30-year - old primigravida patient exposed to seasonal influenza ( h1n1 ) . a ) intervillous ( maternal ) spaces with clusters / sheets of histiocytes ( chronic intervillositis ) and fibrotic fetal chorionic villi with hofbauer cells histiocytic inflammation ( hematoxylin and eosin stain , original magnification 200 ) . b ) dual - stained immunofluorescent assay showing antibodies to influenza a virus ( h1n1 ) ( tetramethylrhodamine isothiocyanate , red ) and cytokeratin ( fluorescein isothiocyanate , green ) in intravillous ( fetal ) and intervillous ( maternal ) space . chronic intervillositis was evident in the placental histology and has been previously described with nonspecific changes in miscarriage and recurrent pregnancy loss ( 4 ) . histiocytes identified from the maternal intervillous space and fetal chorionic villi demonstrated characteristics of viral production ( figure 2 ) . several well - formed viral capsids were noted within the cytoplasm , each containing regularly spaced projections along the surface of the virion corresponding to the hemagglutinin and neuraminidase spikes . tissue sample from 30-year - old primigravida patient exposed to seasonal influenza ( h1n1 ) . electron microscopy ( original magnification 70,000 ) of maternal intervillous space and fetal chorionic villi , showing intranuclear viral transcription aligning along the nuclear envelope electron hypodensities ( asterisks ) , and intracytoplasmic viral production in varying stages shown by numerous electron densities ( v and ^ ) . pcr for m1 capsid protein confirmed influenza a. immunohistochemical testing was performed on tissue from the placenta and on a sampling of fetal organs by using influenza a virus ( h1n1)specific antibody . in the placenta , stippled immunofluorescence ( figure 1 , panel b ) was noted in the histiocytes of the intervillous space ( maternal ) and fetal intravillous histiocytes . in the fetal organs , immunofluorescence for subtype h1n1 was notable in the surface epithelial cells and scattered luminal histiocytes of the fetal respiratory and gastrointestinal tracts . no immunofluorescence was noted within the fetal organs , which implied transamnionic passage of virus ( amnionic fluid infection ) . y - specific immunofluorescent stain with histiocyte counterstaining was performed . because only the intravillous ( fetal ) histiocytes stained positively , maternal and fetal inflammatory responses were confirmed . harris reported higher rates of miscarriage after exposure and infection in the earliest months of pregnancy in the spanish influenza outbreak of 1918 ( subtype h1n1 ) ( 5 ) . hardy et al . reported a similar observation following the asian influenza outbreak of 195758 ( 6 ) , as did investigators in the united kingdom during the 198586 influenza season ( 7 ) . yawn et al . documented influenza a transplacental transfer from the mother to amniotic fluid and fetal heart ( 8) . . summarized placental findings associated with influenza infections in publications from 1987 and 1994 , describing perivillous and villous inflammation with concurrent amniotic fluid immunofluorescence for influenza virus ( 9 ) . in another study ( 10 ) , researchers from beijing , people s republic of china , reported autopsy findings for 2 patients ( 1 of whom was 4 months pregnant ) who died of avian influenza a ( h5n1 ) . viral antigens were present in the maternal pulmonary system , and virus was detected in placental hofbauer cells ( histiocytes ) , cytotrophoblast , fetal lung , circulating mononuclear cells , and liver macrophages ( 10 ) . their documentation of tropism of the influenza virus to placental tissue is similar to the histopathologic features in our report . our unexpected discovery of viral production in placental , maternal , and fetal histiocytes is another example of transplacental passage of influenza . adverse fetal outcomes have reportedly included congenital malformation and schizophrenia , but factors that determine these effects remain unclear ( 11,12 ) . fetal consequences of influenza exposure may be an effect of systemic inflammatory response to the infection , which could represent direct action of the virus on the placenta , the maternal fetal interface , or the fetus . reported the inflammatory response of decidual and fetal tissue in the presence of influenza virus infection and demonstrated expression of mrna for a set of proinflammatory cytokines such as interleukin-6 , tumor necrosis factor- , and granulocyte macrophage colony - stimulating factor , secreted in substantial amounts in response to exposure to influenza virus in vitro ( 13 ) . as our case suggests , a complex interaction of maternal and fetal responses at the interface is a more likely explanation . a total of 15%20% of pregnancies end in spontaneous miscarriage , most before gestational week 12 ( 3 ) . approximately 50% of miscarriages are associated with chromosomal defects , which leaves many other miscarriages unexplained ( 14 ) . examined first trimester miscarriages not associated with chromosomal abnormality , observing histologic changes of chronic intervillositis and increased perivillous fibrin ( 15 ) . the mechanism of loss was thought to be related to a maternal inflammatory response to infectious antigen or an autoimmune phenomenon . whether these findings can be extrapolated to second trimester losses histopathologic features of these early second trimester miscarriages are not well studied , and descriptions are usually included in reports of fetal losses during 1427 weeks , which include iufd ( > 20 weeks ) and miscarriage . consequently , a direct causal relationship between influenza a and fetal demise has not been well established . in our report , a retrospective investigation of the patient s products of conception with ultrastructural , immunohistochemical , and molecular methods that identified subtype h1n1 implies an exposure during her first trimester . type - specific serologic analysis was not possible because she received influenza vaccine several weeks after presumed exposure . the maternal and fetal inflammatory responses ( chronic intervillositis ) after placental infection may have reduced villous surface area for oxygen and nutrient transport , resulting in growth restriction and ultimately second trimester loss . investigation of the effects of influenza in early pregnancy is needed to evaluate potential pathophysiological relationships between maternal exposure , infection , and fetal loss or adverse outcome . during the 200910 influenza pandemic , the centers for disease control and prevention ( atlanta , ga , usa ) collected information from populations exposed to and infected with pandemic ( h1n1 ) 2009 virus . we recommend that future influenza outbreak evaluations of women exposed to influenza with first or early second trimester pregnancy loss include collections of placental and fetal tissue samples . trauma and especially multiple trauma including traumatic brain injury ( tbi ) are the most common killers in children and adults before age 50 around the world [ 13 ] . mortality of multiple trauma including tbi following traffic accidents ranges between 18% and 25% [ 1 , 4 ] . but not only the first hours and days are critical ; for years , more and more multiple trauma patients have been surviving the initial accident and acute care period but only to develop systemic inflammatory response syndrome ( sirs ) , sepsis , or multiple organ dysfunction syndrome ( mods ) in the course of the few weeks of critical care [ 2 , 5 , 6 ] . for 5000 multiple trauma patients in germany , the german trauma registry showed an in - hospital mortality of 12% in 2010 . in the same report , critical care complications such as sirs , sepsis , and mods are reported for 15% of patients . other groups report similar rates of sirs , sepsis , and mods [ 2 , 6 , 7 ] . it is generally known that these complications again deteriorate the prognosis and outcome of the initially surviving trauma patient and increase late mortality to up to 50% if manifest mods is developed [ 2 , 7 , 8 ] . to understand pathophysiological processes leading to multiple organ failure , sirs , and sepsis and taking place during this devastating illness , profound pathophysiological knowledge on the effects of the initial trauma ( first hit ) is essential to minimize additional harm by the following surgical or critical care treatment ( potential second hit ) [ 911 ] . therefore , numerous neurotrauma as well as shock research studies with many different animal and clinical models showed inflammatory changes after trauma [ 1214 ] . only recently , semple et al . reported on the important role of ccl2 in c57bl / j6 mice , peaking at 4 to 12 h following closed head injury and leading to improved functional outcome and lesion size reduction as well as less secondary brain damage . another group showed that the substance minozac may attenuate the effect of proinflammatory cytokines after closed tbi in mice . in the same study , the authors discussed if minozac may also increase long - term neurofunction after tbi . on the other hand , femur fracture and shock in mice are known to trigger an inflammatory response for years , and recently several groups discussed therapeutic strategies to modulate this inflammatory response [ 12 , 14 ] . however , little attention has been paid to the immune reaction following combined trauma and the interaction of the brain and the other organs of the body . this is even more important , since the injury pattern of closed tbi , lower extremity fracture , and shock is very common in multiply injured patients treated in trauma centers around the world . therefore , we investigated the humoral and cellular inflammatory changes of the combined tbi , shock , and femur fracture in a polytrauma model of the mouse . the study was approved by the animal research committee of the hannover medical school ( medizinische hochschule hannover , mhh ) and the county government of lower saxony ( bezirksregierung niedersachsen ) , no . 03/672 . 45 male c57bl / j6 mice aged 810 weeks with a mean weight of 27.4 g ( 3.7 g ) were used for the study . all animals were handled at room temperature for 7 days before treatment . throughout the study period , analgetic therapy during the whole time of the study was provided by metamizol 25 mg/1000 ml ( novaminsulfon ratiopharm , ratiopharm gmbh , ulm ) in the drinking water of the animals ad libitum . all surgical and trauma procedures were performed at a level of deep surgical anesthesia during maintained spontaneous breathing . for anesthesia , subcutaneous injection of ketamine ( ketanest , parke - davis gmbh , karlsruhe , germany ) with a dose of 100 mg / kg body weight and 2% xylazine ( rompun , bayer vital gmbh , leverkusen , germany ) with a dose of 16 the eyes of the animals were covered by dexpanthenol ( bepanthen , hoffmann - la roche ag , grenzach - wyhlen , switzerland ) . 30 of the animals underwent a standard wd - tbi following a model published previously by shapira et al . with modification according to chen et al . . under a surgical level of anesthesia , the animal was positioned in a stereotaxic frame , the head was shaved , and the exposed scalp disinfected by softasept n ( braun , melsungen , germany ) . next , the 3 mm diameter probe was positioned on the planned impact point at the left of the sagittal suture and caudad to the coronal suture . the trauma was now performed with a mean velocity of 3 m / s by a software - guided process ( labview , national instruments , austin , tx , usa ) . afterwards , the animals remained under continuous observation and warming until full recovery was reached . femur fracture was applied by a blunt guillotine device according to van griensven et al . , we used a weight of 400 g falling out of a height of 160 mm on the fixed femur . success of the fracture mechanism was controlled clinically ( crepitation and instability ) , while the animal was still in anesthesia . the guillotine procedure resulted in an a - type femur fracture combined with a moderate soft tissue injury . after completion of the injury , we performed a closed reduction and splinting of the fracture by a piece of wood in proper length that was tied to the fractured extremity proximally and distally of the fracture . after both traumata were set , we draw 60% of the blood volume of the respective mouse via an orbital puncture with a heparinized capillary . volume was restored after 1 hour of shock with ringer 's solution four times the extracted blood volume into the tail vein . this resulted in an individual resuscitation procedure . only after this resuscitation was completed and the animal showed 36c of body temperature , anesthesia was terminated and the animal awoke . subjects were sacrificed 96 hours after trauma by exsanguination in deep surgical anaesthesia , while blood samples for cytokines were extracted . lymphocytes were analyzed using standard flow cytometry ( facs analysis ) for cd-4 , cd-8 , cd-4-cd-8 , and cd-56 cells ( facs calibur , bd biosciences , heidelberg , germany ) . serum cytokines ( tumor necrosis factor , tnf ; chemokine cc2 ligand , ccl2 ; interleukins 6 and 12 , il-6 and il-12 ) were analyzed by cytometric bead array technique ( bdtm cytometric bead array mouse inflammation kit , bd biosciences , pharmingen , germany ) and facs as described earlier . results are reported as mean standard error of the mean ( sem ) . for statistical analysis , the polytraumatized animals showed a significantly higher mortality than the animals of both singular trauma groups ( figure 1 ) . tnf , ccl2 , and il-6 showed significantly higher concentrations at 96 h after trauma in the co - tx group , while the single - trauma groups seem to raise their cytokine levels clearly less and without significant differences between groups wd - tbi and fx - sh ( figure 2 ) . il-12 showed no difference in plasma concentration between the groups at 96 h after trauma . the cd-8 ratio was significantly lower , and the cd-56 ratio was significantly higher in the fx - sh mice than in each of the other groups . while the intracranial effects of traumatic brain injury have been a subject of multiple studies , the aim of this study was to investigate the interaction of the brain injury and systemic shock after lower extremity trauma in regard to changes in the inflammatory response . our main results were : increased levels of tnf , ccl2 , and il-6 in the co - tx group , lower rates of cd-8 and higher rates of cd-56 cells in the fx - sh group , an increased mortality in the combined trauma animal group . however , some weaknesses of our study have to be kept in mind . as always in animal studies , nonetheless , mouse models are very common due to their availability , ease of handling , and practicability . anyhow , a correlation with similar large animal models and additional studies in humans are required to complete our findings and value the clinical relevance . furthermore , the injury pattern may seem artificial in its constellation since one huge challenge of human polytrauma studies is the diversity of injury patters requiring complex inclusion and exclusion procedures to gain comparable populations in terms of injury severity and injury pattern . however , in our point of view , this strengthens our study : the injury pattern and severity of our individuals is highly standardized , and the model and both its components are robust and reproducible . so comparability of our injuries and populations is granted , even more so with regard to the complex hypothesis of a combined trauma being more than only the sum of its parts . this holds even true for standard parameters such as weight , size , sex , and age , that usually also affect human studies of polytrauma patients . our first finding supports any injury severity - related changes in inflammatory response reported from humans [ 2 , 2022 ] . our co - tx group simply suffered more severe injuries than the fx - sh and wd - tbi groups . looking at each monotrauma , our plasma cytokine levels are comparable to those reported earlier in literature [ 12 , 14 ] . however , the levels of the proinflammatory cytokines are elevated not only by factor 2 but by factors 3 to 10 , respectively , even as late as 96 hours after trauma . likewise , in a murine trauma - haemorrhage model , other authors reported tnf elevations [ 23 , 24 ] . the same holds true for tbi in humans [ 25 , 26 ] and clinically relevant models of tbi . while these groups reported elevated tnf levels 1 to 4 h after trauma , later publications also found tnf effects on neuronal recovery and prevention of secondary brain damage . so , while the importance of tnf after peripheral or brain injury remains rather clear and reciprocal effects were described to some extent , we found no earlier report on the extent of tnf plasma level rise after combined tbi and trauma haemorrhage . reported a 3 - 4 fold increase in circulating plasma levels after combined fracture and fluid percussion tbi in rats . in humans , il-6 was classified as the most important secondary cytokine in trauma , not only because of its multitude of mainly proinflammatory effects and its association to the severity of trauma , but also because of its prognostic relevance differing survivors and nonsurvivors . so , highest il-6 levels in the most severe trauma group and the highest mortality are in line with earlier results in literature . similarly , ccl-2 is increased in the brain after tbi and leads to increased migration of intravascular leukocytes across a damaged blood - brain barrier [ 31 , 32 ] . as well ccl-2 was found to play a key role in the development of organ dysfunction after trauma by activating monocytes / macrophages to migrate from the intravascular space into peripheral tissues . while thibeault et al . showed no association between il-6 and ccl-2 after trauma , they could show such an association between tnf and ccl-2 . so , while being in line with recent literature , our findings also remain conclusive within themselves with respect to the inflammatory cascades described earlier . with respect to proinflammatory il-12 , our nonsignificantly low serum levels are backed up by recent literature . in tbi , one author described differences in il-12 after 24 h but not later . in human multiple trauma patients suffering from chest trauma , furthermore , il-12 seems to exert some major effects via cd56 + -(nk- ) cells . however , depressed il-12 production in trauma patients was associated with a shift towards a cd4+(th2 ) type pattern of adverse clinical outcome . while numerous reports focus on intravascular lymphocyte populations and function after trauma , the populations crucial for mods to develop are those in peripheral tissues . the significant decrease of the cd-8 + ratio in the splenic suspension of our fx - sh group is supported by the earlier finding of a decreased ratio of cd-8 + lymphocytes in peripheral tissues after trauma hemorrhage compared to controls . however , we found no report on lymphocyte populations in peripheral tissues after traumatic brain injury . yet , the wd - tbi group might be comparable to controls for peripheral trauma and hemorrhage ( our fx - sh group ) and therefore shows higher cd-8 + ratios . still , this hypothesis does not explain the cd-8 + ratio of the co - tx group . restoration of the low cd-8 + ratio after fx - sh by wd - tbi might be speculated on but so far no theory on responsible mechanisms is known . since this result was rather surprising for our group , detailed evaluation of key processes for lymphocyte populations after combined neuro- and peripheral trauma and hemorrhage must be left for future experiments . cd-56-(nk- ) cells were found in a significantly higher concentration in the splenic suspension of our fx - sh group compared to our wd - tbi and co - tx groups . while the same reasons as above for the physiologic cd-56 ratio in our wd - tbi group may be discussed , in the co - tx group one more aspect needs consideration : raised tnf levels in a murine sepsis model were associated with an increased ratio of nk cells [ 35 , 36 ] . however , for cd-56 + cells as well as for cd-8 + cells , the detailed mechanisms and reasons underlying the differences found in our study can not readily be explained . further experiments should focus on lymphocyte populations in peripheral tissues after tbi and combined tbi and trauma hemorrhage . mortality may just reflect the inflammatory changes and/or trauma load [ 1 , 12 ] . in humans , injury severity clearly is associated with mortality . likewise , injury severity was associated with an increased inflammatory response as measured by increased il-6 levels . so was mortality , an increased il-6 level in multiply injured patients was associated with increased mortality . il-6 even was suggested to be used for prognosis in severe trauma [ 2 , 30 ] . in murine models , other groups support the findings in humans and of our study [ 12 , 14 , 37 ] . our animal model of combined extremity fracture , shock , and closed traumatic brain injury clearly showed that the inflammatory response of the combined trauma is severalfold elevated as compared to the respective monotrauma in terms of humoral parameters . the almost physiological findings for peripheral tissue lymphocyte populations in the isolated tbi and in the combined trauma group as compared to the significantly elevated ratios of the trauma - hemorrhage group are not readily explained and need further evaluation . mortality in our combined trauma model is clearly associated with trauma load and inflammation and such parallels the findings in multiple trauma patients .

a second trimester fetal demise followed influenza - like illness in early pregnancy . influenza a virus ( h1n1 ) was identified in maternal and fetal tissue , confirming transplacental passage . these findings suggested a causal relationship between early exposure and fetal demise . management of future influenza outbreaks should include evaluation of products of conception associated with fetal loss . objective . despite broad research in neurotrauma and shock , little is known on systemic inflammatory effects of the clinically most relevant combined polytrauma . experimental investigation in an animal model may provide relevant insight for therapeutic strategies . we describe the effects of a combined injury with respect to lymphocyte population and cytokine activation . methods . 45 male c57bl/6j mice ( mean weight 27 g ) were anesthetized with ketamine / xylazine . animals were subjected to a weight drop closed traumatic brain injury ( wd - tbi ) , a femoral fracture and hemorrhagic shock ( fx - sh ) . animals were subdivided into wd - tbi , fx - sh and combined trauma ( co - tx ) groups . subjects were sacrificed at 96 h. blood was analysed for cytokines and by flow cytometry for lymphocyte populations . results . mortality was 8% , 13% and 47% for fx - sh , wd - tbi and co - tx groups ( p < 0.05 ) . tnf ( 11/13/139 for fx - sh / wd - tbi / co - tx ; p < 0.05 ) , ccl2 ( 78/96/227 ; p < 0.05 ) and il-6 ( 16/48/281 ; p = 0.05 ) showed significant increases in the co - tx group . lymphocyte populations results for fx - sh , wd - tbi and co - tx were : cd-4 ( 31/21/22 ; p = n.s . ) , cd-8 ( 7/28/34 , p < 0.05 ) , cd-4-cd-8 ( 11/12/18 ; p = n.s . ) , cd-56 ( 36/7/8 ; p < 0.05 ) . conclusion . this study shows that a combination of closed tbi and femur - fracture/ shock results in an increase of the humoral inflammation . more attention to combined injury models in inflammation research is indicated .

acute pancreatitis due to antipsychotic treatment is rare but sometimes causes a fatal adverse effect . some atypical antipsychotic agents , including clozapine , olanzapine , quetiapine , and risperidone , are associated with acute pancreatitis.1,2 ) among them , acute pancreatitis caused by risperidone is the rarest.3,4 ) although most cases of acute pancreatitis due to atypical antipsychotic agents occur within 6 months of starting antipsychotic administration,1 ) we experienced a schizophrenic patient suffering from pancreatitis after more than 6 months of risperidone therapy . a 69-year - old japanese woman was diagnosed with schizophrenia at the age of 30 years and received outpatient care at another mental hospital . her positive symptoms were not prominent , but her cognitive level was so impaired that she could not regulate her appetite and consumed about 2,000 kcal / day in addition to three ordinary meals . she had never smoked , did not drink alcohol , and did not take any illegal drugs . blood tests ( table 1 ) , abdominal ultrasonography , and a computed tomography ( ct ) scan were performed . clinical features were accompanied by laboratory findings of hyperamylasemia ( amylase , 1,191 u / l ) , hyperlipasemia ( lipase , 1,514 u / l ) , and mild liver enzyme elevations . results of the abdominal ultrasonography were positive for gallstones in the gallbladder and distention of the common bile duct . subsequently , the amylase and lipase titers remained high ( 461 u / l and 804 u / l , respectively ) , although alanine and aspartate aminotransferases decreased gradually to normal levels . at this point , we felt that it was safe for her to start taking the risperidone again . two days after starting the risperidone , serum lipase and amylase increased again to 1,275 u / l and 745 u / l , respectively , and ck also increased ( 766 u / l ) . we decided to suspend the risperidone and introduced 10 mg intravenous haloperidol injections once per day . two days after discontinuing the risperidone , the serum amylase decreased ( 605 u / l ) , but the serum lipase level remained elevated ( 1,654 u / l ) . one week after discontinuing the risperidone , the levels of amylase and lipase decreased gradually ( 309 u / l and 542 u / l , respectively ) , and ck dropped to the normal range . as her general clinical condition and biochemical markers were stable , we changed the haloperidol injection to an oral solution of 6 mg / day aripiprazole because her mental condition worsened after stopping the risperidone treatment . her mental status improved with the aripiprazole treatment , and she was discharged without positive laboratory findings . the patient 's monthly blood tests continue to be normal , including amylase , lipase , and blood cell counts . although atypical antipsychotic - induced pancreatitis has been reported in conjunction with hyperglycemia,5 ) the pathophysiological mechanism of these adverse events remains unclear . most antipsychotic - induced pancreatitis occurs within 6 months after administration1 ) ; however , our case developed pancreatitis more than 6 months after the start of risperidone treatment . risperidone is a 5-ht2a antagonist and ameliorates diet - induced necrotic pancreatitis in mice,6 ) and reduced serum pancreatic amylase levels is observed after endoscopic retrograde cholangiopancreatography.7 ) however , there is no evidence of an association between risperidone treatment and acute pancreatitis . a thorough evaluation for pancreatitis , such as alcohol , tumor , and autoimmune causes , gallstones were present , which were due to an adverse effect of risperidone because the two separate risperidone administrations elevated serum amylase and lipase independently . aripiprazole is currently used in such cases , as aripiprazole is thought to have fewer effects on metabolism , including saccharometabolism , than other atypical antipsychotic agents . lifestyle was also a risk factor in this case . thus , it is necessary to monitor pancreatic function in addition to hyperglycemia in such cases . polycystic ovary syndrome ( pcos ) , the most common female endocrine disorder , is a heterogeneous endocrine and metabolic disorder , affecting 6 - 10% of women of reproductive age ( 1 ) . features of pcos may manifest at any age , ranging from childhood ( premature puberty ) , teenage years ( hirsutism , menstrual abnormalities ) , early adulthood and middle life ( infertility , glucose intolerance ) to later life ( diabetes mellitus and cardiovascular diseases ) ( 2 ) . several of these features increase the risk of cardiovascular diseases ( cvd ) in women ( 3 ) and the prevalence of hypertension in women with pcos is about 40% in comparison with a prevalence of about 25.8 in the general population ( 4 ) . pcos is also associated with a higher risk of myocardial infarction ( relative risk ) ( 5 ) and with a compromised cardiovascular profile independent from obesity in young women ( 6 ) . hyperandrogenism and insulin resistance or deficiency was linked to pcos , as early as 1921 , when achard and thiers published their classic description of a bearded woman with diabetes ( 7 ) . the polycystic ovary syndrome was then called the stein - leventhal syndrome , which was first described in 1935 . originally , diagnosis required pathognomonic ovarian findings and the clinical triad of hirsutism , amenorrhea and obesity ( 8) . experimental induction of a polycystic ovarian syndrome ( pcos ) in rodents by brower in 1996 was made possible by the use of a single intramuscular ( i.m . ) the rats ceased ovulation and developed characteristics of human pcos , including large cystic follicles in the ovaries and altered concentrations of luteinizing hormone ( 9 ) . roman chamomile or chamaemelum nobile ( l. ) , ( synonym anthemis nobilis l. from asteraceae family ) , is a perennial herb cultivated in western europe and north africa . in traditional medicine , chamomile flowers are used as an anti - spasmolytic and anti - inflammatory tea for stomach disorders . in women , the antispasmodic effects of chamomile ease menstrual cramps and lessen the possibility of premature labor . chamomile extract 's stimulating effect on leukocytes ( macrophages and b lymphocytes ) is used in skin irritations and eczema ( 11 ) . the tranquilizer/ sedative effects of chamomile depress the central nervous system making it useful for curing insomnia ( 12 ) . studies on chamomile extract have shown that both lipophilic and hydrophilic compounds take part in its therapeutic activity . the most characteristic constituents of this plant species are volatile oil , sesquiterpene lactones and phenols including flavonoids . the main constituents of chamomile flowers include several phenolic compounds , primarily the flavonoids , apigenin , quercetin , patuletin , luteolin and their glucosides . flavonoids are chemical phenylbenzopyrones , which are usually conjugated with sugars and are present in all vascular plants . the benzopyranonring system is a molecular scaffold which is found in flavonoid natural products and has weak aromatase inhibitory activity ( 13 ) . several biological properties have been ascribed to flavonoids , which among them are the wellknown anti - inflammatory , antioxidant , antihepatotoxic and antiviral activities together with their vasculo - protector and spasmolitic effects . one of the 6 major subgroups of flavenoids is the subgroup of flavone including flavon , apigenin and luteolin , which all three exist in chamomile . the effects of flavonoids on the central nervous system have been considered just in the past 10 years . in particular , the studies performed by medina 1989 have demonstrated the capacity of some flavonoids for binding to the central type benzodiazepine ( bzd ) receptors ( 14 ) . apigenin , one of the predominant flavonoids in chamomile , was found to competitively inhibit the binding of flunitrazepam ( a benzodiazepine receptor blocker of ) . at doses up to 30 mg / kg , apigenin was shown to have a clear anxiolytic activity without the sedative or muscle relaxing effects noted for benzodiazepines ( e.g. , valium ) without noticeable anticonvulsant properties ( 14 ) . effects of phytoestrogens on dna synthesis and tyrosine kinase activity show that chrysin , quercetin , apigenin and luteolin inhibit estradiol ( e2)-induced dna synthesis or have antiproliferative properties ( antimutagenic ) , growth inhibitory properties ( 15 ) and inhibitory effects on aromatase activity ( 13 ) . due attention to pcos which is the most common female endocrine disorder in women of reproductive age and the therapeutic activity of chamomile ( flavonoids ) , we saught to ( 1 ) compare the circulating levels of gonadotropins and gonadal steroids before and after injection of chamomile extract in pco induced rats ( 2 ) , study the signs of pcos in the ovarian tissue and ( 3 ) change in the number of dominant follicles upon chamomile extract administration . thirty virgin adult cycling wistar rats , weighting 200 - 220 g were divided into two groups and housed every six mice into a cage under standard conditions ( 212c , 12-hour light/ 12-hour dark cycles ) for at least one week before and throughout the study , with free access to standard chow and tap water ad libitum . the study and all the procedures were carried out in accordance with the guidelines for the care and use of laboratory animals of tehran university of medical sciences ethics committee and that of the national institutes of health guide for care and use of laboratory animals ( publication no . estrous cyclicity was monitored by vaginal smears obtained between 0800 and 1200 hours , and assessed by light microscopy for the relative proportion of leukocytes , epithelial and cornified cells found in daily vaginal lavages , which characteristically change during different stages of the estrous cycle . the rat estrous cycle ( estrus , diestrus 1 , diestrus 2 , and proestrus ) usually lasts about 4 days , in both control and pco rats ( 16 ) . chamomile flowers were collected from natural resources in ahvaz , iran . after grinding the dried flowers , after one week of acclimatization , 8-week - old rats ( n = 30 ) were divided into two groups of control and pco rats . the control group received 0.2 ml corn oil and all the rates assigned to the pco group received an i.m . injection of 0.2 mg estradiol valerate ( ev ) ( aburaihan co. , iran ) , in 0.2 ml of corn oil , to induce pco as described by brawer 1996 ( 9 ) . all the ev - treated rats were evaluated 60 days after the injection , when follicular cysts are first detected . subsequently , pco rats were subdivided into four groups : one group did not receive chamomile extract and other three groups received different doses ( 25 , 50 and 75 mg / kg ) of chamomile alcoholic - extract intraperitoneally for ten days . blood samples were collected and serum luteinizing hormone ( lh ) , follicular stimulation hormone ( fsh ) and estradiol levels were determined by elisa method . , costa mesa , u.s.a . ) , and estradiol kit ( drg international , gmbh , u.s.a . ) . ovaries of the controls and ev - treated rats were removed , cleaned from adherent fat and connective tissue , and fixed in 10% formaldehyde buffer for at least 24 hours . statistical analysis was done by using spss software , version 13 ( spss inc , illinois , u.s.a . ) . comparisons between the controls and ev - treated rats were made by anova , followed by the student - newman - keuls post - hoc test . thirty virgin adult cycling wistar rats , weighting 200 - 220 g were divided into two groups and housed every six mice into a cage under standard conditions ( 212c , 12-hour light/ 12-hour dark cycles ) for at least one week before and throughout the study , with free access to standard chow and tap water ad libitum . the study and all the procedures were carried out in accordance with the guidelines for the care and use of laboratory animals of tehran university of medical sciences ethics committee and that of the national institutes of health guide for care and use of laboratory animals ( publication no . thirty virgin adult cycling wistar rats , weighting 200 - 220 g were divided into two groups and housed every six mice into a cage under standard conditions ( 212c , 12-hour light/ 12-hour dark cycles ) for at least one week before and throughout the study , with free access to standard chow and tap water ad libitum . the study and all the procedures were carried out in accordance with the guidelines for the care and use of laboratory animals of tehran university of medical sciences ethics committee and that of the national institutes of health guide for care and use of laboratory animals ( publication no . estrous cyclicity was monitored by vaginal smears obtained between 0800 and 1200 hours , and assessed by light microscopy for the relative proportion of leukocytes , epithelial and cornified cells found in daily vaginal lavages , which characteristically change during different stages of the estrous cycle . the rat estrous cycle ( estrus , diestrus 1 , diestrus 2 , and proestrus ) usually lasts about 4 days , in both control and pco rats ( 16 ) . chamomile flowers were collected from natural resources in ahvaz , iran . after grinding the dried flowers , after one week of acclimatization , 8-week - old rats ( n = 30 ) were divided into two groups of control and pco rats . the control group received 0.2 ml corn oil and all the rates assigned to the pco group received an i.m . injection of 0.2 mg estradiol valerate ( ev ) ( aburaihan co. , iran ) , in 0.2 ml of corn oil , to induce pco as described by brawer 1996 ( 9 ) . all the ev - treated rats were evaluated 60 days after the injection , when follicular cysts are first detected . subsequently , pco rats were subdivided into four groups : one group did not receive chamomile extract and other three groups received different doses ( 25 , 50 and 75 mg / kg ) of chamomile alcoholic - extract intraperitoneally for ten days . blood samples were collected and serum luteinizing hormone ( lh ) , follicular stimulation hormone ( fsh ) and estradiol levels were determined by elisa method . , costa mesa , u.s.a . ) , and estradiol kit ( drg international , gmbh , u.s.a . ) . ovaries of the controls and ev - treated rats were removed , cleaned from adherent fat and connective tissue , and fixed in 10% formaldehyde buffer for at least 24 hours . statistical analysis was done by using spss software , version 13 ( spss inc , illinois , u.s.a . ) . comparisons between the controls and ev - treated rats were made by anova , followed by the student - newman - keuls post - hoc test . a p - value less than 0.05 was considered statistically significant . estrous cyclicity was monitored by vaginal smears obtained between 0800 and 1200 hours , and assessed by light microscopy for the relative proportion of leukocytes , epithelial and cornified cells found in daily vaginal lavages , which characteristically change during different stages of the estrous cycle . the rat estrous cycle ( estrus , diestrus 1 , diestrus 2 , and proestrus ) usually lasts about 4 days , in both control and pco rats ( 16 ) . chamomile flowers were collected from natural resources in ahvaz , iran . after grinding the dried flowers , after one week of acclimatization , 8-week - old rats ( n = 30 ) were divided into two groups of control and pco rats . the control group received 0.2 ml corn oil and all the rates assigned to the pco group received an i.m . injection of 0.2 mg estradiol valerate ( ev ) ( aburaihan co. , iran ) , in 0.2 ml of corn oil , to induce pco as described by brawer 1996 ( 9 ) . all the ev - treated rats were evaluated 60 days after the injection , when follicular cysts are first detected . subsequently , pco rats were subdivided into four groups : one group did not receive chamomile extract and other three groups received different doses ( 25 , 50 and 75 mg / kg ) of chamomile alcoholic - extract intraperitoneally for ten days . blood samples were collected and serum luteinizing hormone ( lh ) , follicular stimulation hormone ( fsh ) and estradiol levels were determined by elisa method . , costa mesa , u.s.a . ) , and estradiol kit ( drg international , gmbh , u.s.a . ) . ovaries of the controls and ev - treated rats were removed , cleaned from adherent fat and connective tissue , and fixed in 10% formaldehyde buffer for at least 24 hours . statistical analysis was done by using spss software , version 13 ( spss inc , illinois , u.s.a . ) . comparisons between the controls and ev - treated rats were made by anova , followed by the student - newman - keuls post - hoc test . in the present study , we examined the effects of dried chamomile flowers alcoholic extract on the ovaries and uteri of pco - induced rats ( figure 1 ) . the ovary . in the ovarian tissue , the cysts were mainly appeared by a single intramuscular dose of estradiol valerate , 2 mg in 0.2 ml of corn oil rats with pcos which had been treated by 50 mg / kg of chamomile extract showed recovery demonstrated by macroscopic and microscopic morphological examination in the ovarian and uterine tissues . in the ovarian tissue , the cysts had mainly disappeared ( figure 2 ) and the number of dominant follicles had increased ( figure 3 ) and better endometrial tissue arrangements were observable ( figure 4 ) . mean hormonal changes in pco - induced animals , injected with 50 mg / kg of chamomile extract , showed statistically significant differences in comparison with the controls ( p < 0.05 ) , ( table 1 ) . serum levels of estradiol and gonadotropins , lh and fsh , were significantly decreased in the chamomile group relevant to the control group ( p < 0.05 ) . the cysts were mainly disappeared by chamomile administration . in uterus , better endometrial tissue arrangements were also observed upon chamomile administration a schematic illustration of how gaba neurons are involved in the regulation of lh surge . an inhibitory gaba tone induced by estrogen is disrupted by the circadian clock information carried by avp neurons . this results in the disinhibition of gnrh neurons by inhibitory gaba neurons , and in turn it induces the surge of gnrh secretion . experimental polycystic ovary ( pco ) in rodents resembling some aspects of human pco syndrome , for example changes in serum levels of gonadotropin - releasing hormones ( gnrh ) and appearance of cysts , was induced by injecting a long - acting estradiol valerate ( ev ) . classical neuroendocrinological studies indicate that in female rats , the neuronal component responsible for the induction of the lh surge is located in the preoptic area ( poa ) ( 17 , 18 ) . in fact , it has been reported that gonadotropine releasing hormone neurons in the poa express the immediate early gene , c - fos , at the time of the lh surge suggesting that such gnrh neurons in the poa are responsible for the generation of the gnrh surge ( 19 ) . to understand the mechanism underlying the generation of the lh surge , ( i.e. , the gnrh surge ) accumulating evidence suggests that gamma amino butyric acid ( gabaergic ) regulation of gnrh neurons is profoundly involved in the regulation of lh surge ( 20 , 21 ) , ( figure 4 ) . it has been suggested that a decrease in the inhibitory tone of gabaa on gnrh neurons causes lh to surge ( 22 ) . consistently , kimura ( 1994 ) showed that intravenous infusion of bicuculline , a gabaa receptor antagonist , on the morning of proestrus induced a premature surge - like secretion of lh ( 23 ) . studies on chamomile extract have shown that both lipophilic and hydrophilic compounds take part in its therapeutic activity . the most characteristic constituents of this plant species are volatile oil , sesquiterpene lactones and phenolics including flavonoids . the effects of flavonoids on the central nervous system have been considered only in the past 10 years . particularly , the studies performed by medina 1989 have demonstrated the capacity of some flavonoids for binding to the central type benzodiazepine ( bzd ) receptors . apigenin , one of the predominant flavonoids in chamomile , was found to competitively inhibit the binding of flunitrazepam , a benzodiazepine derivative , and was fitted into a pharmacophore model for ligands binding to the gabaa receptor benzodiazepine site . this reflected the affinities of the compounds in the [ ( 3 ) h]-flumazenil binding assay ( 14 , 2426 ) . therefore , the effects of chamomile extract in rats which were shown in the present study can be attributed to the direct activation of the central benzodiazepine site . extract of dried matricaria chamomilla l. flowers not only can induce recovery from an induced pco state in rats , which is probably due to the interaction of the gaba system combined with the effects of chamomile in the regulation of lh surge secretion , it but also can increase dominant follicles . in the uterus , it causes better endometrial tissue arrangements .

acute pancreatitis with antipsychotic treatment is rare but sometimes causes a fatal adverse effect . most cases of acute pancreatitis due to atypical antipsychotic agents are reported to occur within six months of starting antipsychotic administration . acute pancreatitis caused by risperidone is rare . the patient had a high fever , stomachache and vomiting . the results of the abdominal computed tomograhpy scan were negative . the results of the abdominal ultrasonography were positive for gallstones in gallbladder and distention of the common bile duct . she had been fasting and received antibiotic intravenous injections . amylase and lipase titers were high . after risperidone discontinuation , both the levels of the amylase and the lipase were gradually decreased . three months later , the patient still maintains a good clinical balance . although atypical antipsychotic - induced pancreatitis has been reported in conjunction with hyperglycemia , the pathophysiologic mechanism of these adverse events remains unclear . this case got pancreatitis 6 month after risperidone treatment . using the antipsychotic agents , it is necessary to monitor pancreas function . introductionpolycystic ovary syndrome ( pcos ) is a complex endocrine and metabolic disorder associated with ovulatory dysfunction . presently , little is known about the primary factors that initiate pcos . chamomile flowers are used in alternative medicine for its anti - spasmolytic and anti - inflammatory effects . antispasmodic properties of chamomile ease menstrual cramps and lessen the possibility of premature labor . this medicinal herb also stimulates menstruation . in this study , we evaluated the effects of chamomile alcoholic - extract on the biochemical and clinical parameters in a rat model of pcos.materials and methodsestrous cyclicity of 30 virgin adult cycling rats was monitored by vaginal smears obtained between 0800 and 1200 hours . after about 4 days , each rat received an i.m . injection of estradiol valerate ( aburaihan co. , iran ) , 2 mg in 0.2 ml of corn oil , to induce pco . corn oil was injected to the rats in the control group . all the rats in the experimental group were evaluated for follicular cysts 60 days after the injection . rats with pcos were treated by multiple doses ( 25 , 50 , 75 mg / kg ) of intraperitoneal injections of chamomile alcoholic - extract for ten days . the data were statistically analyzed at a significance level of p<0.05 by anova , followed by the student newman - keuls post hoc test.resultsthe histological and hormonal results showed that chamomile can decrease the signs of pcos in the ovarian tissue and help lh secretion in rats ( p<0.05).conclusionthe alcoholic - extract of dried matricaria chamomilla l. flowers can not only induce recovery from a pco induced state in rats , but also increase dominant follicles . additionally better endometrial tissue arrangements can be regarded as another therapeutic effect of chamomile .

phylloquinone , also referred to as vitamin k1 , is a compound present in all photosynthetic plants ( 1 ) . phylloquinone is the primary dietary source of vitamin k. in general , green , leafy vegetables contain the highest known phylloquinone concentrations and contribute approximately 60% of total phylloquinone intake ( 2 , 3 ) . as indicated in table 1 , spinach and collards , which have concomitant high concentrations of chlorophyll associated with the photosynthetic process , hence , dark leaf color have substantially higher concentrations of phylloquinone compared to the more commonly consumed iceberg lettuce , which is substantially paler , hence , lower chlorophyll concentrations . the other plant sources of phylloquinone are certain plant oils including soybean , canola ( also known as rapeseed ) , cottonseed , and olive ( table 1 ) . margarine , spreads , and salad dressings derived from these plant oils are important dietary sources of phylloquinone ( 4 , 5 ) . plant oils are used for preparation of multiple mixed dishes , hence many commercially prepared foods including baked goods also contain small amounts of phylloquinone . although multiple databases now exist that contain some phylloquinone contents of foods ( 6 , 7 ) , the most extensive analysis of phylloquinone in common foods using established food sampling protocols ( 8) are found in the united states department of agriculture ( usda ) nutrient database for standard reference ( http://www.ars.usda.gov/sp2userfiles/place/12354500/data/sr22/nutrilistsr22a430.pdf ; accessed 05.22.10 ) . however , numbers in this table are indicative of median concentrations of all analysis for a given food in the author 's laboratory and may differ from reference because of the inclusion of unpublished data . the commercial hydrogenation of phylloquinone - rich oils results in a transformation of phylloquinone into a hydrogenated form , 2,3-dihydrophylloquinone ( 9 ) . as expected , trans fatty acid concentrations are highly correlated with dihydrophylloquinone concentrations ( 10 ) . in the us food supply commercial hydrogenation of plant oils was a common practice that prolonged shelf life of the oil - based products . many foods sold in fast - food restaurants and frozen prepared products such as fast - food french fries , doughnuts , and breaded fish sticks contained high concentrations of 2,3-dihydrophylloquinone ( 5 ) ( table 1 ) . however , with current controversy regarding the health consequences of trans fatty acid intake ( 11 ) , there have been regional trans fat bans in the us food supply . it is anticipated that the decrease in hydrogenation of plant oils will also reduce the presence of dihydrophylloquinone in the us food supply , hence monitoring of this form of vitamin k in the food supply is warranted . unlike trans fatty acids , dihydrophylloquinone is only present in hydrogenated plant oils , hence monitoring this form of vitamin k in the food supply is a robust approach to monitoring the practice of commercial hydrogenation of plant oils . menaquinones are the other category of vitamin k present in the food supply ( fig . menaquinones are often referred to as vitamin k2 , which is somewhat misleading given that all menaquinones are not alike in their origin or their function . menaquinones are primarily of bacterial origin , and differ in structure from phylloquinone in their 3-substituted lipophilic side chain . the major menaquinones contain 410 repeating isoprenoid units indicated by mk-4 to mk-10 ; forms up to 13 isoprenoid groups have been identified . 1 ) is unique among the menaquinones in that it is not of bacterial origin . instead , mk-4 is formed by a realkylation step from menadione present in animal feeds or is the product of tissue - specific conversion directly from dietary phylloquinone ( 12 , 13 ) . in the united states , menadione is the synthetic form of vitamin k used in poultry feed . as such , mk-4 formed from menadione is present in poultry products in the us food supply ( 14 ) . however , mk-4 formed from phylloquinone is limited to organs not commonly consumed in the diet including kidney . the exceptions are dairy products with mk-4 found in milk , butter , and cheese , albeit in modest amounts . therefore it is unlikely that mk-4 is an important dietary source of vitamin k in food supplies that do not use menadione for poultry feed nor are rich in dairy products . there is growing interest in the health benefits of longer - chain menaquinones , which are limited to certain foods in the food supply . menaquinone-7 ( mk-7 ) is primarily the product of fermentation using bacillus subtilis natto and is present in a traditional japanese soybean - based product called natto . natto contains approximately 2.5 times more mk-7 compared to the phylloquinone content of spinach ( table 1 ) . natto also contains mk-8 and phylloquinone ( 84 and 35 g/100 g , respectively ) , although both are modest in concentration compared to mk-7 ( 6 ) . some cheeses also contain mk-8 and mk-9 ( 6 ) , but these are dependent on cheese production practices , hence the food composition databases are limited in their ability to characterize menaquinone intake across different food supplies . phylloquinone , also referred to as vitamin k1 , is a compound present in all photosynthetic plants ( 1 ) . phylloquinone is the primary dietary source of vitamin k. in general , green , leafy vegetables contain the highest known phylloquinone concentrations and contribute approximately 60% of total phylloquinone intake ( 2 , 3 ) . as indicated in table 1 , spinach and collards , which have concomitant high concentrations of chlorophyll associated with the photosynthetic process , hence , dark leaf color have substantially higher concentrations of phylloquinone compared to the more commonly consumed iceberg lettuce , which is substantially paler , hence , lower chlorophyll concentrations . the other plant sources of phylloquinone are certain plant oils including soybean , canola ( also known as rapeseed ) , cottonseed , and olive ( table 1 ) . margarine , spreads , and salad dressings derived from these plant oils are important dietary sources of phylloquinone ( 4 , 5 ) . plant oils are used for preparation of multiple mixed dishes , hence many commercially prepared foods including baked goods also contain small amounts of phylloquinone . although multiple databases now exist that contain some phylloquinone contents of foods ( 6 , 7 ) , the most extensive analysis of phylloquinone in common foods using established food sampling protocols ( 8) are found in the united states department of agriculture ( usda ) nutrient database for standard reference ( http://www.ars.usda.gov/sp2userfiles/place/12354500/data/sr22/nutrilistsr22a430.pdf ; accessed 05.22.10 ) . however , numbers in this table are indicative of median concentrations of all analysis for a given food in the author 's laboratory and may differ from reference because of the inclusion of unpublished data . the commercial hydrogenation of phylloquinone - rich oils results in a transformation of phylloquinone into a hydrogenated form , 2,3-dihydrophylloquinone ( 9 ) . as expected , trans fatty acid concentrations are highly correlated with dihydrophylloquinone concentrations ( 10 ) . in the us food supply commercial hydrogenation of plant oils was a common practice that prolonged shelf life of the oil - based products . many foods sold in fast - food restaurants and frozen prepared products such as fast - food french fries , doughnuts , and breaded fish sticks contained high concentrations of 2,3-dihydrophylloquinone ( 5 ) ( table 1 ) . however , with current controversy regarding the health consequences of trans fatty acid intake ( 11 ) , there have been regional trans fat bans in the us food supply . it is anticipated that the decrease in hydrogenation of plant oils will also reduce the presence of dihydrophylloquinone in the us food supply , hence monitoring of this form of vitamin k in the food supply is warranted . unlike trans fatty acids , dihydrophylloquinone is only present in hydrogenated plant oils , hence monitoring this form of vitamin k in the food supply is a robust approach to monitoring the practice of commercial hydrogenation of plant oils . menaquinones are the other category of vitamin k present in the food supply ( fig . menaquinones are often referred to as vitamin k2 , which is somewhat misleading given that all menaquinones are not alike in their origin or their function . menaquinones are primarily of bacterial origin , and differ in structure from phylloquinone in their 3-substituted lipophilic side chain . the major menaquinones contain 410 repeating isoprenoid units indicated by mk-4 to mk-10 ; forms up to 13 isoprenoid groups have been identified . 1 ) is unique among the menaquinones in that it is not of bacterial origin . instead , mk-4 is formed by a realkylation step from menadione present in animal feeds or is the product of tissue - specific conversion directly from dietary phylloquinone ( 12 , 13 ) . in the united states , menadione is the synthetic form of vitamin k used in poultry feed . as such , mk-4 formed from menadione is present in poultry products in the us food supply ( 14 ) . however , mk-4 formed from phylloquinone is limited to organs not commonly consumed in the diet including kidney . the exceptions are dairy products with mk-4 found in milk , butter , and cheese , albeit in modest amounts . therefore it is unlikely that mk-4 is an important dietary source of vitamin k in food supplies that do not use menadione for poultry feed nor are rich in dairy products . there is growing interest in the health benefits of longer - chain menaquinones , which are limited to certain foods in the food supply . menaquinone-7 ( mk-7 ) is primarily the product of fermentation using bacillus subtilis natto and is present in a traditional japanese soybean - based product called natto . natto contains approximately 2.5 times more mk-7 compared to the phylloquinone content of spinach ( table 1 ) . natto also contains mk-8 and phylloquinone ( 84 and 35 g/100 g , respectively ) , although both are modest in concentration compared to mk-7 ( 6 ) . some cheeses also contain mk-8 and mk-9 ( 6 ) , but these are dependent on cheese production practices , hence the food composition databases are limited in their ability to characterize menaquinone intake across different food supplies . the current us dietary guidelines for intakes of vitamin k are 90 and 120 g / day for women and men , respectively ( 15 ) . these guidelines are termed adequate intakes ( ai ) because the institute of medicine concluded in 2001 that there were insufficient data available to generate a precise recommendation for vitamin k. the ai values for vitamin k were generated from the third national health and nutrition examination survey ( nhanes iii , 19881994 ) and based on the median phylloquinone intake in the united states for each age and gender category ( 15 ) . in the absence of abnormal bleeding associated with low vitamin k intakes among adults , it was assumed that the current intakes are adequate . however , the adequacy of intake defined by an absence of bleeding is controversial . furthermore , the elderly report median intakes below the current ai for adults . as reviewed elsewhere ( 16 ) , there is controversy regarding biochemical measures of subclinical vitamin k deficiency and as a consequence , the true dietary requirement of vitamin k is unknown . for the purpose of this review , dietary intakes of vitamin k will be presented relative to the current ai . by comparison , the guidelines in the united kingdom are 1 g / kg body / day ( 17 ) and are set at 75 g / day for adult men , 60 g / day for women , aged 1829 year , and 65 g / day for women 30 years and over in japan ( 18 ) . estimates of phylloquinone intake in various populations are probably more accurate than intakes of other forms of vitamin k and there is a substantial difference in the reported intakes from a number of countries , which seems to be related to food consumption practices in different areas ( 1823 ) . for example , the mean reported vitamin k intake of young japanese women ( mean 21.2 years , n=124 ) was about 230 g / day ( 18 ) . the estimated phylloquinone intakes in northern china , england , and scotland were 247 , 103 , and 70 g / day , respectively ( 23 , 24 ) . of note , phylloquinone intakes have been decreasing over the last two decades in the united kingdom , consistent with a concomitant decline in leafy green vegetable consumption ( 25 ) . based on the nhanes iii data , the elderly ( defined as older as 71 years of age ) have median phylloquinone intakes lower than the current ai ( 89 and 79 g / day for women and men , respectively ) . this observation supports the findings of others who have reported very low phylloquinone intakes among those in nursing homes ( 26 ) and those with alzheimer 's disease ( 27 ) . given emerging association of low vitamin k intakes with risk of certain diseases such as cardiovascular disease and osteoarthritis ( 16 ) , the elderly present a potentially vulnerable subgroup of the population with respect to vitamin k. very little is known about the contribution of dietary menaquinones to overall vitamin k nutrition and although it has been stated that approximately 50% of the daily requirement for vitamin k is supplied by the gut flora through the production of menaquinones , there is little evidence to support this estimate ( 28 ) . in one study among adults with acute bacterial overgrowth as induced by omeprazole , menaquinones produced by these bacteria had some contribution to vitamin k status during dietary phylloquinone restriction , but not enough to restore biochemical measures of vitamin back to normal range ( 29 ) . as previously indicated , there are regional differences in the forms and content of menaquinones in the food supply . for example , natto is unique to a traditional japanese diet whereas the cheeses that contain high concentrations of mk-8 and mk-9 appear to be most prevalent in european dairy producing food supplies . although there are reported menaquinone intakes , these are limited to studies from japan ( 18 ) and the netherlands ( 21 ) , and are low compared to phylloquinone intakes . in the united states , menaquinones are limited in the food supply and have not been systematically assessed . there are surprisingly little data on the relative biological availability of different forms of vitamin k among different food sources . furthermore , there is a growing body of literature to suggest that our understanding of vitamin k is still rudimentary in light of new developments regarding different forms . as previously stated , phylloquinone is of plant origin , with absolute intakes being predominantly from green leafy vegetables . phylloquinone is tightly bound to the membranes of plant chloroplasts , and is less bioavailable compared to phylloquinone obtained from plant oils and/or dietary supplements ( 30 ) . some estimates place the absorption of phylloquinone to be 10% from plants compared to supplements ( 31 ) . however there appear to be differences in absorption compared to the plant species , with phylloquinone obtained from broccoli and collards having greater absorption compared to spinach ( 31 , 32 ) . similarly , and not unexpected because vitamin k is lipophilic , addition of a fat source to the meal results in higher absorption . for a more comprehensive review of vitamin k absorption phylloquinone differs from 2,3-dihydrophylloquinone by a saturation of a single bond at the 2,3 position of the side chain ( 9 ) . surprisingly this single substitution results in a lower absorption of dihydophylloquinone compared to an equimolar amount of phylloquinone ( 34 ) . there is also indirect evidence of lower activity of dihydrophylloquinone as an enzyme cofactor , which currently is the only known function of vitamin k. the implications of this poor bioavailability and activity are currently unknown , although at least one study suggests a detrimental effect on bone mineral density among older adults in a community - based cohort ( 35 ) . the potential impact of poor utilization of dihydrophylloquinone on bone health will be of little importance in the future should hydrogenated oils be removed from the food supply . mk-7 , when administered in the form of natto in equimolar amounts to phylloquinone administered in the form of spinach , has a peak height difference of more than 10-fold compared to phylloquinone , with a half - life of 56 hours , compared to 7.5 hours for phylloquinone ( 36 ) . whereas all forms of vitamin k appear to be initially associated with triglyceride - rich lipoproteins ( trl ) , the longer chain menaquinones including mk-7 and mk-9 are also associated with low - density lipoprotein ( ldl ) . these preliminary data suggest that the menaquinones have different transport pathways and distribution , which has implications for transport to extra - hepatic tissue such as bone ( 33 ) . emerging studies on mk-4 challenge our current understanding of vitamin k. as demonstrated using stable isotopes , mk-4 is a conversion product of phylloquinone via the intermediate , menadione ( 37 ) . there appears to be both local and systemic conversion to mk-4 , with the local conversion being the predominant pathway . to add complexity to the interpretation is the observation that this conversion does not occur in all tissues . whereas the liver contains primarily phylloquinone and very long - chain menaquinones , mk-4 is the predominant form in the brain , pancreas , and glands ( 38 ) . in terms of dietary intakes , mk-4 intakes are low compared to other forms of vitamin k. however , dietary phylloquinone converts to mk-4 in those tissues where mk-4 appears to be required so it is likely that low mk-4 intakes are of little consequence to health when there is adequate dietary phylloquinone available for conversion to mk-4 . in conclusion , much of our understanding of vitamin k nutrition has focused on the primary dietary source , phylloquinone . in contrast , the menaquinones are present in the food supply , but there are limited food composition data available . as a corollary , estimates of intakes of menaquinones are very limited and our understanding of their role in vitamin k nutrition is not well understood . based upon work supported by the us department of agriculture , agricultural research service under cooperative agreement no . any opinions , findings , conclusions , or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the us department of agriculture . adult articular cartilage has insufficient ability to repair after either erosion or damage and it is unlikely to be reformed to normal condition once it has been impaired . articular cartilage repair has been based on finding and resolving the primary reason for the injury , but it has been recognized that only treating the damage site is not adequate to gain good long - term functionality . the matrix environment of hyaline cartilage is different from that of subchondral bone in biological , physical and chemical characteristics . this led to the proposal that in order to gain positive results in tissue repair , the microenvironment in which the repair process takes place should be retouched , such as those elements that play a pro - angiogenic , conductive , cell differentiation and chemotactic role . this tendency has been the reason behind a growth in using genetic and tissue engineering in the examination of articular cartilage repair . a number of natural and artificial biomaterials have been used as scaffolds for cartilage reformation , but their biological effectiveness as well as safety remains unresolved . therefore , scaffold biomaterials extracted from the patients own body have long been a tempting choice . platelet - rich plasma ( prp ) , which can be easily harvested from autologous blood , has been used in clinical experiments for decades . prp is rich in growth factors , containing those that help the proliferation of chondrogenic cells and discharging of cartilaginous matrix , such as platelet - derived growth factor ( pdgf ) , transforming growth factor ( tgf - b ) , vascular endothelial growth factor ( vegf ) , basic fibroblast growth factor ( bfgf ) and insulin - like growth factor ( igf ) . a new member in platelet concentrated products is platelet - rich fibrin ( prf ) , which can be prepared easily without biochemical blood handling . prf is similar to prp except in biochemical architectures because of slow polymerization during prf preparation . such a network leads to a more competent cell mobilization and repopulation and consequently healing . stromal cell - derived factor-1 ( sdf1 ) , a chemokine factor expressed by bmscs , regulates the trafficking , homing and migration of stem cell by mediating chemokinesis and chemotaxis into the injury site . we believe that autologous prp and prf could be used alone as a 3-d scaffold capable of cartilage tissue regeneration because of releasing endogenous growth factors . sdf1 stimulates cartilage mesenchymal stem cells ( c - mscs ) , synovial membrane mesenchymal stem cells ( sm - mscs ) , and bone marrow stromal cells ( bmscs ) to migrate to the subchondral bone in full - thickness defects to make cartilage . the aim of the study was to evaluate tissue repair along with prp and prf or without treatment . the secondary objective was to evaluate the efficacy of prp and prf used with sdf1 . the 9 ml of whole blood collected in a tube containing 1 ml an anticoagulant ( 3.8% sodium citrate solution ) was centrifuged for 5 minutes at 2,000 rpm and the supernatant was placed in a new tube . the plasma at the top was discarded and the remaining 1ml of liquid prp precipitate was collected . after mixing prp ( 200 l / defect ) and 40 mmol / l calcium chloride ( cacl2 ) , a gel was formed . the prp gel bonded to the adjacent cartilage about 10 minutes after implantation . the 10 ml blood samples collected from the hearts of six rabbits without anticoagulant were immediately centrifuged at 3,000 rpm for 10 minutes . in the middle of the tube , a fibrin clot was formed between supernatant acellular plasma and the lower red corpuscles . before transplantation , 600 l of rhsdf1 alpha ( 120 ng / ml peprotech ) was incubated with prp gel , prf and gelatin separately for 2 hours at 37c . a total of 18 adult male white japanese rabbits ( weighing 2.5 - 3.0 kg ) were used in this study . the animals were housed in metal wire cages , in a temperature - controlled room under a 12:12 h light - dark cycle at 2224c and 5060% relative humidity . animal interventional experiments were carried out at animal experiments of tehran university of medical sciences under the rules and regulation of the animal care and use committee of shiraz university school of medicine . the 36 bilateral knees of the 18 rabbits were randomly divided into 6 experimental groups of 6 knees per group . each animal was sedated by intramuscular injection of ketamine hydrochloride 60 mg / kg and xylazine 6 mg / kg . in sterile conditions , a full - thickness cylindrical cartilage defect of 4 mm in diameter and 3 mm in depth was created in the patellar groove using a standard size stainless biopsy punch . i , the defects were left unfilled ( control group ) . in group ii , the defects were filled with platelet - rich plasma only ( prp group ) . in group iii , the defects were filled with platelet - rich fibrin only ( prf group ) . in group iv , the defects were filled with gelatin containing stromal cell - derived factor-1 ( gel+sdf1 group ) . in group v , the defects were filled with platelet - rich plasma containing stromal cell - derived factor-1 ( prp+sdf1 group ) . in group vi , the defects were filled with platelet - rich fibrin containing stromal cell - derived factor-1 ( prf+sdf1 group ) in a randomized manner . the distal parts of the femurs were excised , photographed and graded for cartilage repair , according to the international cartilage repair society score ( icrs ) macroscopic assessment scores ( table 1 ) . icrs macroscopic evaluation of cartilage repair after gross evaluation , samples were fixed in 4% paraformaldehyde for 7 days , decalcified in 10% edta for 3 weeks and then embedded in paraffin and cut perpendicularly into 5 m sections . then , the sections were stained for general histology with hematoxylin and eosin ( h&e ) and with toluidine blue to estimate the cartilaginous matrix distribution . the re - created tissue was scored blindly according to the icrs scale ( table 2 ) . icrs visual histological assessment scale immunofluorescent analysis was performed using a monoclonal antibody against collagen ii ( cat number : ma1 - 37493 ) . heat - induced epitope retrieval was done in citrate buffer ( sodium citrate , 10 mm ; ph 6.0 ) in a pressure cooker for 4 minutes at full pressure . subsequent to cooling , endogenous peroxidase was blocked using a 3% hydrogen peroxide solution for 20 minutes . the slides were then washed with phosphate buffer solution ( 10 mm ; ph 7.4 ) and incubated with primary antibodies overnight at 4c . the slides were washed again three times with pbs and subsequently incubated with an fitc - conjugated secondary antibody ( cat number : 11555100 ) according to the respective manufacturer s instructions . total rna extraction was performed using rnx - plus kit ( cinnagen ) according to the protocol . the concentration and quantification of total rna were measured with spectrophotometer , with the od260/od280 ratio of all rna samples 1.92.0 and od260/od230 ratio up to 2 . the first strand cdna was synthesized with the first - strand cdna synthesis kit ( bioneer kit , k-2101 ) . for each reaction , 1 g ( 1 l ) rna was used for reverse transcription , in a mixture of 20 pmoles ( 1 l ) random primer , and 18 l depc - d.w with a final volume of 20 l . the mixture was incubated at 15c for 1 min , 50c for 60 minutes and heated at 95c for 5 minutes to terminate the reaction . qpcr was performed in a volume 1 l of primer and 1 l from template also add 3 l depc.d.w with 5 l master mix ( accupower 2x greenstartmqpcr master mix , bioneer kit ) . all pcr reactions were in the following condition : initial 95c for 15 minutes , followed by 35 cycles at 95c for 15 s and 60c for 30 s. after completing the qpcr , their products run on agarose gels for quality check and real - time data analyzed by livak rules . the sequences of primers used in real - time pcr analyses were as follows : aggrecan forward gagaccaagtcctcaagccc ; reverse ctctgtctcctggcaggttc . a comparison between groups for each outcome was carried out with the kruskal - wallis test . the 9 ml of whole blood collected in a tube containing 1 ml an anticoagulant ( 3.8% sodium citrate solution ) was centrifuged for 5 minutes at 2,000 rpm and the supernatant was placed in a new tube . the plasma at the top was discarded and the remaining 1ml of liquid prp precipitate was collected . after mixing prp ( 200 l / defect ) and 40 mmol / l calcium chloride ( cacl2 ) , a gel was formed . the prp gel bonded to the adjacent cartilage about 10 minutes after implantation . the 10 ml blood samples collected from the hearts of six rabbits without anticoagulant were immediately centrifuged at 3,000 rpm for 10 minutes . in the middle of the tube , a fibrin clot was formed between supernatant acellular plasma and the lower red corpuscles . before transplantation , 600 l of rhsdf1 alpha ( 120 ng / ml peprotech ) was incubated with prp gel , prf and gelatin separately for 2 hours at 37c . a total of 18 adult male white japanese rabbits ( weighing 2.5 - 3.0 kg ) were used in this study . the animals were housed in metal wire cages , in a temperature - controlled room under a 12:12 h light - dark cycle at 2224c and 5060% relative humidity . animal interventional experiments were carried out at animal experiments of tehran university of medical sciences under the rules and regulation of the animal care and use committee of shiraz university school of medicine . the 36 bilateral knees of the 18 rabbits were randomly divided into 6 experimental groups of 6 knees per group . each animal was sedated by intramuscular injection of ketamine hydrochloride 60 mg / kg and xylazine 6 mg / kg . in sterile conditions , a medial para - patellar arthrotomy was made in both knees . a full - thickness cylindrical cartilage defect of 4 mm in diameter and 3 mm in depth was created in the patellar groove using a standard size stainless biopsy punch . ii , the defects were filled with platelet - rich plasma only ( prp group ) . in group iii , the defects were filled with platelet - rich fibrin only ( prf group ) . in group iv , the defects were filled with gelatin containing stromal cell - derived factor-1 ( gel+sdf1 group ) . in group v , the defects were filled with platelet - rich plasma containing stromal cell - derived factor-1 ( prp+sdf1 group ) . in group vi , the defects were filled with platelet - rich fibrin containing stromal cell - derived factor-1 ( prf+sdf1 group ) in a randomized manner . the distal parts of the femurs were excised , photographed and graded for cartilage repair , according to the international cartilage repair society score ( icrs ) macroscopic assessment scores ( table 1 ) . after gross evaluation , samples were fixed in 4% paraformaldehyde for 7 days , decalcified in 10% edta for 3 weeks and then embedded in paraffin and cut perpendicularly into 5 m sections . then , the sections were stained for general histology with hematoxylin and eosin ( h&e ) and with toluidine blue to estimate the cartilaginous matrix distribution . the re - created tissue was scored blindly according to the icrs scale ( table 2 ) . icrs visual histological assessment scale immunofluorescent analysis was performed using a monoclonal antibody against collagen ii ( cat number : ma1 - 37493 ) . heat - induced epitope retrieval was done in citrate buffer ( sodium citrate , 10 mm ; ph 6.0 ) in a pressure cooker for 4 minutes at full pressure . subsequent to cooling , endogenous peroxidase was blocked using a 3% hydrogen peroxide solution for 20 minutes . the slides were then washed with phosphate buffer solution ( 10 mm ; ph 7.4 ) and incubated with primary antibodies overnight at 4c . the slides were washed again three times with pbs and subsequently incubated with an fitc - conjugated secondary antibody ( cat number : 11555100 ) according to the respective manufacturer s instructions . total rna extraction was performed using rnx - plus kit ( cinnagen ) according to the protocol . the concentration and quantification of total rna were measured with spectrophotometer , with the od260/od280 ratio of all rna samples 1.92.0 and od260/od230 ratio up to 2 . the first strand cdna was synthesized with the first - strand cdna synthesis kit ( bioneer kit , k-2101 ) . for each reaction , 1 g ( 1 l ) rna was used for reverse transcription , in a mixture of 20 pmoles ( 1 l ) random primer , and 18 l depc - d.w with a final volume of 20 l . the mixture was incubated at 15c for 1 min , 50c for 60 minutes and heated at 95c for 5 minutes to terminate the reaction . qpcr was performed in a volume 1 l of primer and 1 l from template also add 3 l depc.d.w with 5 l master mix ( accupower 2x greenstartmqpcr master mix , bioneer kit ) . all pcr reactions were in the following condition : initial 95c for 15 minutes , followed by 35 cycles at 95c for 15 s and 60c for 30 s. after completing the qpcr , their products run on agarose gels for quality check and real - time data analyzed by livak rules . the sequences of primers used in real - time pcr analyses were as follows : aggrecan forward gagaccaagtcctcaagccc ; reverse ctctgtctcctggcaggttc . a comparison between groups for each outcome was carried out with the kruskal - wallis test . the macroscopic evaluation of the treated areas in the prf+sdf1 group revealed complete repair and integrated well with the surrounding cartilage as compared with other groups ( figure 1 ) . a post - hoc analysis showed that icrs scores of all experimental groups were significantly higher than those in the control group and the scores of the prf+sdf1 group were greater than those of the other groups ( p<0.001 ) ( figure 2 ) . gross appearance of defects in the trochlear groove at four weeks after surgery , respectively . b ) prp group : healing tissue is thinner than normal surrounding cartilage and shows a large fissure . d ) gel+sdf1 group : repaired tissue covers the defect almost completely , but is thin and distinguishable from the surrounding cartilage . e ) prp+sdf1 group : healing tissue covers defect with smooth white tissue and distinguishable from the surrounding cartilage . f ) prf+sdf1 group : repaired tissue covers the defect completely and no obvious margin was notable . a ) icrs macroscopic and b ) icrs microscopic scores in the regenerated cartilage of defects in the trochlear groove at four weeks postoperatively . data are presented as meansd . * significant difference with non - treated control ( p<0.05 ) ; * * significant difference with rpp group ( p<0.05 ) . four weeks after surgery , the regenerated regions were evaluated by h&e and toluidine blue staining . the regenerated regions of the prf+sdf1 group were entirely filled by the repaired tissue and showed better results than the other groups ( figures 3 and 4 ) . a post - hoc analysis showed that icrs scores of all groups , except for the prp group , were significantly higher than the control group ( p<0.001 ) . the scores of the prf+sdf1 group were greater than the other groups , but were not significant when compared with the gel+sdf1 and prp+sdf1 groups . the scores of the prp group were significantly lower than the other groups except for the prf group , which was not significant ( figure 2 ) . the qualitative study of immunofluorescent staining of collagen ii demonstrated that neo - cartilages were minimally positive in the control , prp , and prf groups . in contrast , the gel+sdf1 , prp+sdf1 and prf+sdf1 groups exhibited a remarkable distribution of type ii collagen ( figure 5 ) . the microscopic appearance of h&e staining of defects in the trochlear groove at four weeks after surgery , respectively ( original magnification=200 ) . a ) control group : the defect is concave and a noticeable thin layer of non - cartilaginous tissue . b ) prp group : the defect is filled by fibrous tissue with a crack . d ) gel+sdf1 group and ( e ) prp+sdf1 group : the defects in both groups are filled with a repaired tissue , which are thinner than the normal surrounding cartilage . f ) prf+sdf1 group : the defect is entirely filled by the repaired tissue , resembling the healthy cartilage surrounding the tissue . histological appearance of toluidine blue staining of defects in the trochlear groove at four weeks after surgery , respectively ( original magnification=200 ) . b ) prp group , and c ) prf group : the defects in both groups have poor staining fibrocartilagous tissues and poorly organized ecm . d ) gel+sdf1 group , and e ) prp+sdf1 group : the repaired tissues show a good staining in both groups . a ) control group , b ) prp group , c ) prf group , d ) gel+sdf1 group , e ) prp+sdf1 group , and f ) prf+sdf1 group . the neo - cartilages are minimally positive in a , b and c. the neo - cartilages are positive in d and e. a remarkable distribution of type ii collagen is seen in f. four weeks after surgery , a real - time pcr analysis revealed that the mrna expression of sox9 was significantly greater in the regenerated tissue of all groups than the untreated control group ( p<0.001 ) . the level of gene expression of aggrecan in all groups was significantly higher than the control group , except for the prp group that was not significant ( p<0.001 ) ( figures 6 and 7 ) . the amount of gene expression of sox9 and aggrecan in the regenerated cartilage of defects in the trochlear groove at four weeks postoperatively . data are presented as meansd . * significant difference with non - treated control ( p<0.05 ) . the quality checking of rt - pcr for sox9 and aggrecan from the regenerated cartilage of defects in the trochlear groove at four weeks after surgery . lane a : control group where transplantation was not performed ; lane b : prp group ; lane c : prf group ; lane d : gel+sdf1 group ; lane e : prp+sdf1 group , and lane f : prf+sdf1 group . the macroscopic evaluation of the treated areas in the prf+sdf1 group revealed complete repair and integrated well with the surrounding cartilage as compared with other groups ( figure 1 ) . a post - hoc analysis showed that icrs scores of all experimental groups were significantly higher than those in the control group and the scores of the prf+sdf1 group were greater than those of the other groups ( p<0.001 ) ( figure 2 ) . gross appearance of defects in the trochlear groove at four weeks after surgery , respectively . b ) prp group : healing tissue is thinner than normal surrounding cartilage and shows a large fissure . d ) gel+sdf1 group : repaired tissue covers the defect almost completely , but is thin and distinguishable from the surrounding cartilage . e ) prp+sdf1 group : healing tissue covers defect with smooth white tissue and distinguishable from the surrounding cartilage . f ) prf+sdf1 group : repaired tissue covers the defect completely and no obvious margin was notable . a ) icrs macroscopic and b ) icrs microscopic scores in the regenerated cartilage of defects in the trochlear groove at four weeks postoperatively . data are presented as meansd . * significant difference with non - treated control ( p<0.05 ) ; * * significant difference with rpp group ( p<0.05 ) . four weeks after surgery , the regenerated regions were evaluated by h&e and toluidine blue staining . the regenerated regions of the prf+sdf1 group were entirely filled by the repaired tissue and showed better results than the other groups ( figures 3 and 4 ) . a post - hoc analysis showed that icrs scores of all groups , except for the prp group , were significantly higher than the control group ( p<0.001 ) . the scores of the prf+sdf1 group were greater than the other groups , but were not significant when compared with the gel+sdf1 and prp+sdf1 groups . the scores of the prp group were significantly lower than the other groups except for the prf group , which was not significant ( figure 2 ) . the qualitative study of immunofluorescent staining of collagen ii demonstrated that neo - cartilages were minimally positive in the control , prp , and prf groups . in contrast , the gel+sdf1 , prp+sdf1 and prf+sdf1 groups exhibited a remarkable distribution of type ii collagen ( figure 5 ) . the microscopic appearance of h&e staining of defects in the trochlear groove at four weeks after surgery , respectively ( original magnification=200 ) . a ) control group : the defect is concave and a noticeable thin layer of non - cartilaginous tissue . b ) prp group : the defect is filled by fibrous tissue with a crack . d ) gel+sdf1 group and ( e ) prp+sdf1 group : the defects in both groups are filled with a repaired tissue , which are thinner than the normal surrounding cartilage . f ) prf+sdf1 group : the defect is entirely filled by the repaired tissue , resembling the healthy cartilage surrounding the tissue . histological appearance of toluidine blue staining of defects in the trochlear groove at four weeks after surgery , respectively ( original magnification=200 ) . b ) prp group , and c ) prf group : the defects in both groups have poor staining fibrocartilagous tissues and poorly organized ecm . d ) gel+sdf1 group , and e ) prp+sdf1 group : the repaired tissues show a good staining in both groups . a ) control group , b ) prp group , c ) prf group , d ) gel+sdf1 group , e ) prp+sdf1 group , and f ) prf+sdf1 group . the neo - cartilages are minimally positive in a , b and c. the neo - cartilages are positive in d and e. a remarkable distribution of type ii collagen is seen in f. four weeks after surgery , a real - time pcr analysis revealed that the mrna expression of sox9 was significantly greater in the regenerated tissue of all groups than the untreated control group ( p<0.001 ) . the level of gene expression of aggrecan in all groups was significantly higher than the control group , except for the prp group that was not significant ( p<0.001 ) ( figures 6 and 7 ) . the amount of gene expression of sox9 and aggrecan in the regenerated cartilage of defects in the trochlear groove at four weeks postoperatively . data are presented as meansd . * significant difference with non - treated control ( p<0.05 ) . the quality checking of rt - pcr for sox9 and aggrecan from the regenerated cartilage of defects in the trochlear groove at four weeks after surgery . lane a : control group where transplantation was not performed ; lane b : prp group ; lane c : prf group ; lane d : gel+sdf1 group ; lane e : prp+sdf1 group , and lane f : prf+sdf1 group . this study considered the effects of prp and prf with or without sdf1 in full - thickness lesions in rabbits knees . a literature review indicated that the effects of prf+sdf1 and prp+sdf1 were not studied so far . we demonstrated that the implantation of prf+sdf1 led to the greatest yield scores of full - thickness lesions in rabbits knee . therefore , it can be concluded that the scaffolds containing sdf1 have a better result on cartilage repair . zhang w. et al . implanted a combination of collagen scaffold to mimic the subchondral bone matrix environment containing sdf1 in order to repair partial - thickness defects in rabbits knee . the results demonstrated that collagen scaffold containing sdf1 improved the repairing process by a better support for c - mscs and sm - mscs migration and adhesion six weeks after transplantation . some researchers used ultra - purified alginate gel containing sdf1 to improve osteochondral lesions in rabbits knee . according to their studies , the repairing effect of osteochondral defects is elevated , because sdf1 increases the migration of host cells ( mainly bmscs ) to the site of injury , and this makes sdf1 as a potential factor in cartilage repair . they studied the length of the hypertrophic zone by immunohistochemical analysis of tibia cartilaginous growth plates of chicken embryos , which were incubated with sdf1 for 2 , 4 , and 6 days in organ culture . the results showed the length of the hypertrophic zone was significantly elevated after 4 and 6 days . in injured tissue with the permissive matrix environment , endogenous stem cells can participate in corrective process . some studies have introduced populations of stem / progenitor cells in the synovial membrane and articular cartilage , which show characteristic of stem cells and have the potential for cartilage repair . the findings of the above studies are in agreement with our results that show sdf1 can facilitate the migration of c - mscs and sm - mscs to full - thickness defect sites . although sdf1 has been notified to promote inflammatory cell migration , we could not find inflammatory cells in the newly formed tissue in rpf , prp and gel containing sdf1 groups . in parallel with our data , a study on tendon repair by exogenous sdf1 showed improvement of injury site and diminishing accumulation of inflammatory cells . in prf , the fibrin formation meshes , which are similar to biochemical architectures of the natural one , can trap a significant amount of circulating cytokines . this method increases the lifespan of these cytokines ( as a long - term effect ) , without which , it will be only released and used at the time of the primary phase of matrix remodeling . as a result , when the cells begin extracellular matrix remodeling to reconstruct the injured site , the cytokines will be available in the affected area for the required longer period . cytokines are important factors in the delicate balance of tissue homeostasis . in a pilot study , . showed that all patients with osteochondral defects who received bm - mscs transplanted on platelet - rich fibrin glue ( pr - fg ) had a significant improvement in their functional knee scores and mri findings after 6 months and remained stable 12 months post operation . in agreement with this clinical study , choukroun j. et al . revealed that filling a tooth socket by prf can enhance the healing process without any complication . in a supplementary study , they reported that adding prf to bone allograft in sinus elevation surgery can lead to 4-month reduction in healing time by increasing the speed of bone allograft maturation . however , in contrast to our results , an in - vivo study by shao x.x . et al . on osteochondral defects in rabbits knee showed that bmscs seeded in fibrin glue matrix forms a poor - quality cartilage - like tissue . it can be concluded that the reason for varying results can be due to the differences in biomechanical conditions of defect types . et al . reported that differentiation of bone - marrow derived stem cells can be affected by mechanical signals . moreover , suitable recurring compressive stress significantly increases chondrocyte proliferation in addition to aggrecan and collagen synthesis by chondrocytes . some researchers reported that cell adhesion and migration can facilitate by some elements such as different cell type and substrate stiffness . most importantly , in a separate study , wang h. et al . had compared the amount of bounded bovine chondrocytes on various surfaces of middle deep zone bone or calcified cartilage or hyaline cartilage . they reported that different contact surface stiffness give different ability to cells for migration and attachment . activated prp can be used in different forms like gel as a scaffold , liquid form injection or can be infiltrated in the tissue thickness . the selection of each form relies upon the type of tissue and the existing problem . by degradation of the fibrin skeleton of prp growth factors play a significant role in tissue repair , especially in the regenerative process of tissues with low cellular density and vascularization , such as cartilage . a study showed that if prp was applied in full - thickness chondral lesions , the signs of healing will be observed , but it did not show healthy or functional behavior . wu w. et al . reported cartilage formation into the prp scaffold after implanting prp gel as a carrier for cultured autologous chondrocytes in the subcutaneous tissue of rabbits knees . another study suggested that transplantation of prp gel along with synovial membrane derived mesenchymal stem cells ( sdscs ) can facilitate the repair of osteochondral lesions in rabbits knees . perhaps the underlying reason for different results is various compositions of this plasma fraction in different species , including rabbits . prf was harvested from an autologous blood sample that had a limited volume and prf tissue banks were not achievable . in the present study , in addition to using prp and prf as scaffolds for cell seeding , we used a mixture of sdf1 and prp and prf scaffolds to enhance the migration of host cells . in our research , the effects of prp and prf alone on the full - thickness defects were also studied separately . we found that the added sdf1 stimulated cell or the combination of prp , prf , and sdf1 stimulated the observed improvement .

vitamin k is present in the diet in the forms of phylloquinone and menaquinones . phylloquinone , which is the major dietary source , is concentrated in leafy plants and is the vitamin k form best characterized in terms of food composition and dietary intakes . in contrast , menaquinones are the product of bacterial production or conversion from dietary phylloquinone . food composition databases are limited for menaquinones and their presence in foods varies by region . dietary intakes of all forms of vitamin k vary widely among age groups and population subgroups . similarly , the utilization of vitamin k from different forms and food sources appear to vary , although our understanding of vitamin k is still rudimentary in light of new developments regarding the menaquinones . background : the purpose of this study was to create biomaterial scaffolds like platelet - rich plasma ( prp ) and platelet - rich fibrin ( prf ) containing stromal cell - derived factor-1 ( sdf1 ) as a chemokine to induce hyaline cartilage regeneration of rabbit knee in a full thickness defect.methods:we created a full thickness defect in the trochlear groove of thirty - six bilateral knees of eighteen mature male rabbits . the knees were randomly divided into six groups ( group i : untreated control , group ii : prp , group iii : prf , group iv : gelatin+sdf1 , group v : prp+sdf1 , and group vi : prf+sdf1 ) . after four weeks , the tissue specimens were evaluated by macroscopic examination and histological grading , immunofluorescent staining for collagen type ii , and analyzed for cartilage marker genes by real - time pcr . the data were compared using statistical methods ( spss 20 , kruskal - wallis test , bonferroni post hoc test and p<0.05).results : macroscopic evaluations revealed that international cartilage repair society ( icrs ) scores of the prf+sdf1 group were higher than other groups . microscopic analysis showed that the icrs score of the prp group was significantly lower than other groups . immunofluorescent staining for collagen ii demonstrated a remarkable distribution of type ii collagen in the gel+sdf1 , prp+sdf1 and prf+sdf1 groups compared with other groups . real - time pcr analysis revealed that mrna expression of sox9 and aggrecan were significantly greater in the prf+sdf1 , prp+sdf1 , gel+sdf1 and prf groups than the control group ( p<0.05).conclusion : our results indicate that implantation of prf scaffold containing sdf1 led to the greatest evaluation scores of full - thickness lesions in rabbits .

it was initially developed for angiography , but it has also been found useful in oral and maxillofacial radiology ( omfr ) . the introduction of cbct has made three - dimensional imaging possible in omfr . cbct offers a 3d approach to data acquisition , image reconstruction , image display , and image interpretation . cbct scan can be achieved in a relatively short period of time , making it ideal for imaging children and claustrophobic adults . the patient finds the examination comfortable as it can be performed with the patient in a standing or sitting position . the technique is characterized by fast image acquisition with relatively high three - dimensional resolution , lower radiation exposure to patients , and fewer artifacts when compared with conventional computed tomography ( ct ) . the voxels are isotropic and the resolution varies from 0.07 to 0.25 mm depending on the manufacturer . these values exceed most high - grade multi - slice ct units in terms of spatial resolution , real time image analysis , and three - dimensional display . cbct technology has been used for preoperative implant planning , evaluation of the jaws , and presurgical assessment of the bone volume required for orthognathic surgery . as cbct systems have become more prevalent , its use has gained attention in the field of otolaryngology . cbct of the inner ear [ figures 1 and 2 ] is effective in the diagnoses of most malformations , dysplasia , traumatic lesions , thin osseous labyrinthine wall erosion , or dehiscence . the advantages and diagnostic value of cbct have been demonstrated in the analysis of the position of cochlear implants in the temporal bone , the free visualization of alloplastic middle ear titanium , gold , or platinum implants . it is also possible that the smallest bone structure or position of the prosthesis can be defined with high precision contributing valuable information to the diagnosis , preoperative planning , and prevention of intra - operative complications . 60-year - old female patient with clinical history of hearing loss diagnosed with sensorineural hearing loss . cbct of temporal bone three - dimensional image with air cavity volume rendering ( green ) , shows normal inner ear : semicircular canals ( white arrows ) , vestibule ( white star ) , cochlea ( yellow arrow ) and internal auditory canal ( red star ) . 28-year - old male patient with clinical history of bilateral hearing loss and semicircular canal dehiscence diagnosed with conductive hearing loss due to bilateral fixing of the malleus . bilateral cbct of temporal bone three - dimensional image with osseous volume rendering demonstrates normal superior semicircular canal . a semicircular canal dehiscence ( scd ) is characterized by a pathological opening in a small section of the bony wall of the semicircular canal ( sc ) of the inner ear . an scd can be found in three different places according to different etiologies : the superior [ figures 3 and 4 ] , lateral , and posterior scs . regardless of differences in scd location , dehiscence patients have complained of similar symptoms such as vertigo , oscillopsia , and/or hearing loss . 35-year - old male patient with clinical history of vertigo and autophony diagnosed with left superior semicircular canal dehiscence . cbct of temporal bone ( a and d ) coronal ( b ) sagittal ( c ) oblique sagittal images show a dehiscence of the left superior semicircular canal ( white arrow ) . 17-year - old patient with clinic history of progressive hearing loss , without vertigo and oscillopsia diagnosed with sensorineural hearing loss and right superior semicircular canal dehiscence . cbct of temporal bone , air cavity volume rendering ( green ) images ( a ) sagittal view , ( b ) endocranial view , ( c ) osseous volume rendering oblique sagittal view and ( d ) osseous volume rendering superior view show superior semicircular canal dehiscence ( yellow arrow in a ) , ( yellow arrow in b ) , ( white arrow in c ) , and ( white arrow in d ) . superior semicircular canal dehiscence ( sscd ) syndrome is characterized by sound and/or pressure - induced vertigo and oscillopsia due to a dehiscence of bone overlying the superior sc in the roof of the temporal bone or in the neighborhood of the epidural space . minor et al . , first described this in 1998 , and it has become a new and exciting concept in the field of otology . vertigo as a result of loud noises ( tullio phenomenon ) , autophony , oscillopsia , disequilibrium due to sound changes in middle ear pressure , or changes in intracranial pressure were observed in these patients . sscd may present with a variety of atypical vestibular and/or auditory symptoms , which can be suggestive of otosclerosis . sscd is relatively rare , the reported cases in the literature range from 0.4% to 0.7% with the majority of patients being asymptomatic . several etiologies have been linked to hereditary , developmental , intracranial hypertension , and trauma . other authors have suggested that sscd may be due to a congenital or a genetic condition . sscd most commonly occurs along the apex or the roof of the sc , termed the arcuate eminente . however , approximately 9.1% of sscd may also occur more posteriorly or medially , involving the groove of the superior petrosal sinus [ figure 5 ] . 42-year - old male patient , with clinical history of cranial trauma and initial diagnosis of left temporal bone fracture diagnosed with left temporal bone fracture and ipsilateral superior semicircular canal dehiscence . cbct of temporal bone shows incidental find of dehiscence of the superior semicircular canal ( black arrow ) in superior petrosal sinus canal area ( white arrow ) , cochlea ( white star ) , and ossicle chain ( black star ) . diagnosis of sscd is based on the presence of vestibular or auditory symptoms and radiologic findings . the dehiscence is confirmed with high resolution computed tomography ( hrct ) scanning of the temporal bones using bone window settings . this is a key component in the diagnosis of sscd , especially when a surgical intervention is contemplated . minor reported a sensitivity of 100% , a specificity of 99% , a positive predictive value of 99% , and a negative predictive value of 100% in the identification of sscd . a semicircular canal dehiscence ( scd ) is characterized by a pathological opening in a small section of the bony wall of the semicircular canal ( sc ) of the inner ear . an scd can be found in three different places according to different etiologies : the superior [ figures 3 and 4 ] , lateral , and posterior scs . regardless of differences in scd location , dehiscence patients have complained of similar symptoms such as vertigo , oscillopsia , and/or hearing loss . 35-year - old male patient with clinical history of vertigo and autophony diagnosed with left superior semicircular canal dehiscence . cbct of temporal bone ( a and d ) coronal ( b ) sagittal ( c ) oblique sagittal images show a dehiscence of the left superior semicircular canal ( white arrow ) . 17-year - old patient with clinic history of progressive hearing loss , without vertigo and oscillopsia diagnosed with sensorineural hearing loss and right superior semicircular canal dehiscence . cbct of temporal bone , air cavity volume rendering ( green ) images ( a ) sagittal view , ( b ) endocranial view , ( c ) osseous volume rendering oblique sagittal view and ( d ) osseous volume rendering superior view show superior semicircular canal dehiscence ( yellow arrow in a ) , ( yellow arrow in b ) , ( white arrow in c ) , and ( white arrow in d ) . superior semicircular canal dehiscence ( sscd ) syndrome is characterized by sound and/or pressure - induced vertigo and oscillopsia due to a dehiscence of bone overlying the superior sc in the roof of the temporal bone or in the neighborhood of the epidural space . minor et al . , first described this in 1998 , and it has become a new and exciting concept in the field of otology . vertigo as a result of loud noises ( tullio phenomenon ) , autophony , oscillopsia , disequilibrium due to sound changes in middle ear pressure , or changes in intracranial pressure were observed in these patients . sscd may present with a variety of atypical vestibular and/or auditory symptoms , which can be suggestive of otosclerosis . sscd is relatively rare , the reported cases in the literature range from 0.4% to 0.7% with the majority of patients being asymptomatic . several etiologies have been linked to hereditary , developmental , intracranial hypertension , and trauma . other authors have suggested that sscd may be due to a congenital or a genetic condition . sscd most commonly occurs along the apex or the roof of the sc , termed the arcuate eminente . however , approximately 9.1% of sscd may also occur more posteriorly or medially , involving the groove of the superior petrosal sinus [ figure 5 ] . 42-year - old male patient , with clinical history of cranial trauma and initial diagnosis of left temporal bone fracture diagnosed with left temporal bone fracture and ipsilateral superior semicircular canal dehiscence . cbct of temporal bone shows incidental find of dehiscence of the superior semicircular canal ( black arrow ) in superior petrosal sinus canal area ( white arrow ) , cochlea ( white star ) , and ossicle chain ( black star ) . diagnosis of sscd is based on the presence of vestibular or auditory symptoms and radiologic findings . the dehiscence is confirmed with high resolution computed tomography ( hrct ) scanning of the temporal bones using bone window settings . this is a key component in the diagnosis of sscd , especially when a surgical intervention is contemplated . minor reported a sensitivity of 100% , a specificity of 99% , a positive predictive value of 99% , and a negative predictive value of 100% in the identification of sscd . teymoortash et al . , analyzed radiological imaging of 43 structures of the temporal bone by cbct and ct in 38 human temporal bones . the frequency of visualization of these anatomic structures were studied and statistically analyzed : 9 structures ( 20.1% ) of axial scans and 5 structures ( 11.6% ) of coronal scans could be identified as statistically significantly ( p < 0.05 ) more often in cbct than in ct . they determined that anatomical structures of the temporal bone can be identified significantly better in cbct than in ct scans . a dehiscent - appearing superior canal was seen in 4.0% of cases , while published pathologic studies reported that only 0.5% of temporal bone sscs have a dehiscence ( p < 0.001 ) . they established that the prevalence of dehiscent - appearing superior canal on thin - section temporal bone scanning with reformation in the ssc plane is much higher than anticipated by pathologic studies . the results were compared with intra - operative findings to evaluate the diagnostic value of cbct in cases of erosion of the scs . they determined the presence of a fistula of the scs in 100% of the cases . in conclusion , when compared with multi detector computed tomography , cbct imaging for the inner ear is an excellent alternative . it results in lower radiation to patients , fewer artifacts , and equal or higher resolution depending on the unit used . primary cysts have a lined epithelium and secondary cyst could be secondary to trauma . epidermoid splenic cysts are example of primary congenital cysts that contain an epithelial lining , unlike secondary cysts , which are composed of fibrous tissues , when the cyst is large they have nonspecific abdominal symptoms like pain , nausea , or a palpable mass usually in the left upper quadrant . epidermoid cysts and parasitic cysts are examples of primary cysts and usually have a classic presentation on imaging . despite imaging modalities and the patient s history , it can be difficult to diagnose an epidermoid cyst without a histological examination . the purpose of this paper is to discuss variable and atypical radiological presentation of primary splenic cysts including epidermoid cysts . a 51-year - old female presented to the ed using private transportation with complaints of left upper quadrant abdominal pain , nausea and vomiting for a couple days . she had significant medical history of hypertension , gerd , and hyperlipidemia with a bmi of 37.5 and no previous surgical history . patient had quit smoking cigarettes two years prior , does not drink alcohol or use any illicit drugs . physical exam was significant to being mildly tender to palpation over her left upper quadrant . vitals , complete blood count , basic metabolic panel , liver function tests , and urine analysis were all within normal limits . abdominal ultrasound ( fig . 1 ) visualized a 7.7 cm complex mass in the medial aspect of the spleen . 2 ) revealed a hypodense cystic mass in the medial aspect of the spleen measuring 10.4 cm 8.2 cm with a hounsfield attenuation of 15 ; containing multiple foci of fat with hounsfield attenuation of 102 . patient was assumed to have a confined cystic tumor of unknown etiology without any acute complications at this point . after long discussion with the patient with this large cystic mass of the spleen of unknown pathology , splenectomy was offered to the patient . she underwent uneventful laparoscopic hand - assisted splenectomy 6 weeks after presentation to the ed . she continued to have an uneventful recovery on follow - up with no recurrence of painful symptoms . 4 ) , it weighed 404 g , and was 17 11 5 cm in dimensions and was partially cystic . 11 10 7 cm smooth walled yellow tan cyst was present . on microscopic examination , sections demonstrated unremarkable splenic parenchyma and cyst walls lined by stratified squamous epithelium . immunohistochemical stains demonstrated an adequate distribution of cd20 + b - lymphocytes and cd3 + t - lymphocytes . . examples of primary benign neoplasms involving the spleen include haemangiomas , lymphangiomas , angiomas and splenic mesenchymal tumors such as lipomas , angiomyolipomas , fibromas , fibrosarcomas , and leiomyosarcomas . . they could be a pseudocyst secondary to trauma or a true cyst , which might be infectious or congenital such as an epidermoid cyst . epidermoid splenic cysts are primary congenital cysts that contain an epithelial lining , unlike secondary cysts which are composed of fibrous tissues , . patients usually do not present with specific symptoms if the cyst is small , and when the cyst is large , they have nonspecific abdominal symptoms like pain , nausea , or a palpable mass usually in the left upper quadrant . on imaging , epidermoid and parasitic cysts , which are also primary cysts , can be similar in appearance showing up as well - defined solitary lesions with septations . on computed tomography , septations , calcifications and internal debris . when findings are consistent with multiple neighboring cysts or daughter cysts , parasitic cysts are in favor as a diagnosis . epidermoid cysts of the spleen usually have a thin - walled anechoic appearance on ultrasound with no evidence of calcification on computed tomography . on t2-weighted magnetic resonance imaging , the cyst is hyper - intense , and on t1-weighted imaging , it is hypo - intense but can be increased depending on cyst contents . we used computed tomography to accurately identify the location , size , and adjacent structures of the cyst with an understanding that a definitive diagnosis would only be made by histopathological examination . we identified a complex mass on ultrasound and a hypodense lesion on computed tomography with multiple foci of fat , which were very different demonstrations from what is expected of a classic epidermoid or parasitic cyst on imaging . when examined , an epidermoid cyst usually has a white or grey smooth internal wall with fibrous trabeculations . under microscopic examination , an epithelium is visualized that is stratified squamous , which was consistent with our findings , and the contents can be serous , turbid , or thick and vary in color . surgical treatment is recommended for a symptomatic splenic cyst and for cysts larger than 5 cm , , . marsupialization or fenestration as treatments are indicated for superficial cysts , but they have a high recurrence rate , . in our case a large cyst , greater than 5 cm was identified and extended behind the stomach and into the medial aspect of the spleen . a total splenectomy was our indicated diagnostic and treatment of choice due to size of the cyst , presentation with symptoms , and also as a preventative intervention against future rupture , hemorrhage , infection , or recurrence . it is important to recognize that epidermoid splenic cysts may not always present in a classic fashion on imaging so it is trivial to include them as a differential diagnosis when warranted . the exact nature of a cyst will not be known prior to histological examination as in our case when imaging is not accurate or definitive in differentiation . therefore , the cyst was treated based on symptomology , size , and location and a total splenectomy which was indicated , was performed . bilal kharbutli , md and sharmilee vuyyuru , do have contributed in the study concept and writing .

superior semicircular canal dehiscence is a relatively new syndrome in the field of otology . it is of unknown etiology presenting with a variety of vestibular and auditory symptoms and radiologic findings play a crucial role in its diagnosis . cone beam computed tomography has been shown to be a powerful tool in the field of otolaryngology . it is a three dimensional technique that uses lower radiation resulting in fewer artifacts and offers higher resolution when compared with multi - slice computed tomography . it is considered to be an excellent imaging modality for radiological exploration of the ear . highlightssplenic tumors are not common , many can have minimal to no symptoms , and they can be found incidentally.splenic tumors can be primary or metastatic . they can be benign or malignant.splenic cysts can be infectious , traumatic or congenital.epidermoid splenic cysts may not always present in a classic fashion on imaging , making diagnosis challenging.many splenic masses and complex cysts would require surgical pathological diagnosis .

fungal infections are well - recognized life threatening problems in solid organ transplant ( sot ) , namely in liver transplant recipients . host and environmental factors are critical and important determinants regarding the epidemiology of fungal infections in transplantation . the incidence and aetiology of fungal infections differ dramatically between hematopoietic stem cell transplantation and sot . in the last group , those receiving lung grafts have the highest incidence ( 7.9% ) followed by heart ( 3.4% ) , liver ( 3.1% ) , kidney ( 1.1% ) , and pancreas ( 0.7% ) . in liver transplanted patients , the incidence of invasive fungal infections ranges from 4 to 50% and candida spp . and aspergillus spp fungal cutaneous and subcutaneous infections are frequently associated with people with professional activities related to agriculture or forestry work and the entry of the fungus is facilitated by penetrating injuries caused by thorns , splinters , nails , etc . the limbs are the most affected , but other body parts can also be involved . alternaria general characteristics include the production of dark - coloured phaeodictyospores in chains , and a beak of tapering apical cells [ 6 , 7 ] . some species are recognized to cause cutaneous phaeohyphomycoses and are described as emerging pathogens in immunocompromised patients , being transplantation the most common risk factor and also associated to the use of prednisone . a 65-year - old male caucasian was observed in a dermatology consultation of a central hospital in lisbon on 16th april 2014 . the patient presented with two painless , fast - growing nodules which had developed within 4-months . one of the nodules was located on the right leg and the other one on the dorsum of the left hand ( figs . 1 and 2 ) . he had a clinical history of 6-months liver transplantation and at the time of observation was under immunosuppressive therapy with tacrolimus 8 mg once a day , mycophenolate mofetil 500 mg twice a day and prednisone 20 mg once a day . the patient referred a previous history ( previous to the transplantation ) of agricultural activities with frequent skin injuries . physical examination revealed a well - defined erythematous nodule with 1.4 cm of maximum diameter , with central haematic crusts and a peripheral scali collarette on the dorsum of the left hand and a violaceous nodule with 1.5 cm with central haematic crusts and serous exudate on the right leg . skin biopsies were collected on that day from both nodules using a 4 mm biopsy punch and sent for histological and mycological examination . histological sections of the biopsy were stained with periodic acid - schiff and results retrieved on day + 13 revealed an inflammatory infiltration of the dermis and the presence of multiple fungal spores and hyphae . the mycological analysis of the biopsy material was performed using with calcofluor - white staining , followed by observation under fluorescence microscopy . clinical specimens were cultured on sabouraud 's dextrose agar containing chloramphenicol at 30 c and 35 c . cultures yielded a grey velvety fungus , with a dark - brown reverse on day + 14 . when sub - cultured both on malt extract agar for identification , the microscopic examination of the moulds performed by teased mount and lacto - phenol cotton blue preparation , showed different macroscopic features : green ( leg biopsy ) to grey ( hand biopsy ) colonies both with dark reverse . microscopic examination showed chains of brown conidia with rounded base and beaked apex , with transverse and oblique septation . there were a slight difference between microscopy from leg and hand biopsy , in the first one , conidial chains were more branched than in the second one , and conidia were less tubular ( figs . 3 and 4 ) . from the above microscopic and macroscopic features , the isolates were identified as alternaria spp . on day + 20 and sequenced for speciation . the lesions were excised on day + 34 and the patient started antifungal therapy on the same day with oral itraconazole ( 100 mg daily ) for 3 months with adjustment of immunossupressive therapy . total genomic dna was extracted from purified colonies and the internal transcribed spacer ( its ) region of ribosomal dna ( rdna ) of these isolates was amplified using primer set its1 ( 5tccgtaggtgaacctgcgg3 ) and its4 ( 5tcctccgcttattgatatgc3 ) . sequencing of both strands was performed and nucleotide sequences were edited using the programme chromas2 and aligned using the programme clustal x2 . the sequences obtained were compared with sequences deposited in the genbank and cbs - knaw fungal biodiversity centre databases . molecular sequencing resulted in the identification of alternaria infectoria from the culture obtained from the patient 's hand and alternaria alternata from the culture obtained from the patient 's leg . the sequencing coverage was high ( 99100% ) , as well as the homologies with reference strains ( 98% ) , which is the value considered as an acceptable identification to species level . in fact , we obtained a sequence with a homology of 98% and 99.5% with alternaria infectoria and alternaria alternata , respectively . nucleotide sequences of the isolates were deposited in the genbank database under the accession numbers kp171632 ( alternaria infectoria ) and kp171633 ( alternaria alternata ) . phaeohyphomycosis can be caused by different species , being a. alternata and a. infectoria the most commonly isolated . dematiaceous fungi have melanin in their cell walls , which represent a virulence factor for several pathogenic fungi . the raise of patients under immunosuppressive drug therapy has increased and so the risk of fungal infections is likely to increase . the patient of this case report lives in a rural area , but did not refer trauma after transplantation . however , after a detailed questionnaire , the patient referred that before the transplantation he carried out different agricultural activities and had skin injuries . cutaneous fragility induced by hypercorticism could be an important co - factor permitting direct inoculation from the environment . nevertheless , several fungi can also remain latent for long periods of time and the clinical symptoms of fungal infection may appear several years after the inoculation of the etiological agent . there are currently no standardised treatments for cutaneous infections caused by alternaria spp . in those cases and when it is possible , the reduction of the immunosuppression level can be enough to resolve the lesions , although the long - term use of systemic itraconazole is often recommended . co - existent fungal infections associated with alternaria spp are sometimes referred [ 1723 ] , despite infrequently . furthermore , most of the published cutaneous fungal infection cases refer infection due to only one species as etiological agent . to our knowledge , this is the first case described of a cutaneous co - infection with two different species of alternaria . infection with different species can be under diagnosed , since when multiple lesions are observed , biopsy material is normally taken from only one of them . early detection and appropriate antifungal therapy are essential in the treatment of this emergent infection , molecular identification is a useful tool to distinguish mixed fungal infection . prospective studies have shown the superiority of laparoscopic appendectomy in the treatment of acute appendicitis . however , this technique has not yet gained widespread acceptance because of its longer operative time and lower cost effectiveness when compared with the open approach . moreover , although recent retrospective studies have shown that laparoscopic appendectomy is associated with a significantly shorter hospital stay , other reports demonstrate no significant difference in hospital stay when the laparoscopic and the open approach were compared . the minilaparoscopic approach is associated with less abdominal wall trauma , a lower infection rate at the trocar site and abdominal wall hernia , and produces excellent cosmetic results . in this study , our goal was to confirm the safety and show the feasibility of the minilaparoscopic approach for the treatment of acute appendicitis . between may 1999 and june 2001 , a minilaparoscopic appendectomy was attempted in 37 patients with a preoperative diagnosis of acute appendicitis and without a previous history of abdominal surgeries . the pneumoperitoneum was created by insertion of a veress needle through a small incision in the umbilicus . a 2.2-mm trocar was placed laterally to the left rectus abdominis muscle and served to manipulate a 2.2-mm , 0-degree laparoscope . another 5-mm trocar was placed just below the umbilical crease and was used for the ultrasonic scalpel . a third 2.2-mm trocar was placed suprapubically in the midline and was used for grasper access . exploration of the right lower abdominal cavity identified either an inflamed appendix or ruled out appendicial pathology . in the presence of appendicial pathology , the appendix was held with an atraumatic grasper and dissected with a 5-mm ultrasonic scalpel . an endoloop was positioned at the base of the appendix and tied 1 cm distally to its base . the appendix was then dissected by using an ultrasonic scalpel and placed in a sterile 8 and 1/2-size glove finger used as a specimen bag and removed through the 5-mm trocar site . all specimens were sent to the pathology laboratory for analysis . at the end of the procedure , all trocars were removed and the fascial defect at the 5-mm trocar site was repaired with absorbable sutures . all skin wounds were closed with steri - strips , and sterile dressings were applied . all patients were discharged with written instructions regarding diet , physical activity , use of oral nonsteroidal anti - inflammatory drugs ( nsaids ) for pain control , and follow - up clinic visits were scheduled for 1 week , 1 month , and 6 months postoperatively . the study population consisted of 37 patients , of which 7 were males and 30 were females . the average age of the patients was 26 years ( range , 7 to 48 ) . minilaparoscopic appendectomy was initially attempted in 37 patients ; however , it was only completed in 31 ( 84% ) . in 2 patients ( 5.3% ) , the procedure was completed with the conventional laparoscopic approach , because both patients had a perforated appendix . both patients were operated on with larger trocars and instruments because this made it safer to manipulate and resect the appendix and to wash the abdominal cavity . in 1 patient ( 2.7% ) , the procedure was converted to an open approach through a midline incision due to diffuse fecal peritonitis as a consequence of a ruptured appendix . in 3 patients ( 8% ) , an appendectomy was not performed because of pathology confined to the ovary . one wound infection occurred at the umbilical trocar site , which was treated conservatively . during the extraction of the specimen through the 2.2-mm umbilical trocar , the specimen bag ruptured and the inflamed appendix contaminated the abdominal wall . of the 31 patients , four patients ( 13% ) had a normal appendix , 26 patients ( 84% ) had acute appendicitis , and 1 patient ( 3% ) had a perforated appendix . the average operating time was 34 minutes ( range , 15 to 80 ) , and the median length of stay was 1.2 days ( range , 1 to 5 ) . postoperative pain management consisted of 2 doses of 10 mg of ketorolac tromethamine given intravenously . in all patients , wounds resulting from trocar placement healed well . previous studies have confirmed the superiority of laparoscopic appendectomy in the treatment of acute appendicitis . it is associated with decreased postoperative pain , a shorter length of hospitalization , a faster return to normal activity , an early resumption of dietary intake , and fast resolution of postoperative ileus . however , this technique has not yet gained widespread acceptance because of its longer operative time and higher cost compared with the open approach . in a prospective , randomized study of 50 patients , minn et al showed that the laparoscopic approach did not offer any improved benefits compared with the open approach for the routine patient with acute appendicitis . nevertheless , we believe that the laparoscopic approach allows good exploration of the abdominal cavity and may be helpful in ruling out different pathological states . in our experience , minilaparoscopic exploration of the abdominal cavity diagnosed pathology of the ovary in 8% of patients . it has been shown that 80% of women who underwent an open appendectomy , compared with 10% of women treated with laparoscopic appendectomy , may develop adhesions that in fact may be responsible for the infertility or chronic abdominal pain , or both . based on our experience , the procedure was completed with the conventional laparoscopic approach in 2 patients ( 5.3% ) because of a perforated appendix . we have learned that it was safer to insert bigger trocars and instruments to manipulate and resect the appendix , and also wash the abdominal cavity . we believe that converting to conventional laparoscopy was not a technical regression because the outcome did not differ from the outcome in those who underwent the minilaparoscopic appendectomy . it is generally true that the cost of laparoscopic appendectomy is higher than that of open appendectomy . although shorter hospitalization , less pain medications , and better patient compliance are well - established benefits associated with laparoscopic treatment , the financial burden associated with it in the long - term is also well established . sterile use of finger cut gloves instead of sterile specimen containers and use of nondisposable trocars and instruments may contribute greatly to the reduction in cost . it can be used multiple times ; it is versatile and can be used for other minimally invasive procedures , thus lowering its overall cost . the benefits of the laparoscopic approach in terms of better diagnostic accuracy and safety , especially for women of childbearing age , outweighs the disadvantage of a longer average operative time of about 17 minutes . in terms of advantages associated with the use of the minilaparoscopic approach , a reduction in abdominal wall trauma and a decrease in the number of complications , such as infection of the trocar site and abdominal wall hernia have been reported elsewhere . moreover , the smaller dimension of the trocars accounts for approximately a 70% reduction in postoperative pain , compared with that for the conventional laparoscopic approach . one patient , however , developed a wound infection at the umbilical trocar site that resulted from direct contact of the inflamed appendix with the abdominal wall after rupture of the finger bag . to avoid such a complication in the future , we modified the technique by extracting the bag though the 5-mm suprapubic incision , and since then no similar complications have occurred . the wounds from trocar placement healed well , and the general condition of all patients was excellent at 6-month follow - up . this study shows that the minilaparoscopic approach has the same advantages as the conventional approach in terms of better diagnostic accuracy and safety , especially for women of childbearing age .

fungal invasive infections are rare in general population but are an emergent cause of infection in the immunocompromized population , especially in the solid organ transplant recipients . herein the authors report a clinical case of a liver transplanted patient suffering a cutaneous co - existent infection with a. alternata as well as a. infectoria.to our knowledge this is the first case of cutaneous concomitant infection due to those two species reported not only in portugal but also worldwide . the patient was treated with surgical excision of the lesions and oral itraconazol without relapse . background : minilaparoscopic appendectomy for appendicitis is not a well - established procedure . this approach provides less abdominal wall trauma , fewer complications , and excellent cosmetic results . our aim was to show the feasibility and safety of the minilaparoscopic approach.methods:minilaparoscopic appendectomy was performed in 37 patients . two 2.2-mm trocars were used to manipulate a 2.2-mm , 0-degree laparoscope and for grasper access . a 5-mm trocar was used for the ultrasonic scalpel.results:no deaths occurred . in 3 patients ( 8% ) , appendectomy was aborted due to pathology of the ovary . conversion to the open approach occurred in 2.7% of patients . the average operating time was 34 minutes ( range , 15 to 80 ) , and the median length of hospital stay was 1.2 day ( range , 1 to 5).conclusions : the minilaparoscopic approach a ) has the same advantages as the conventional laparoscopic approach in terms of better diagnostic accuracy and safety ; b ) a low incidence of complications ; and c ) yields excellent cosmetic results .

mental health problems , such as depression and anxiety disorders , are often underrecognized and untreated . . showed that the prospect of being treated increases with the severity of the illness , but also that half of those affected by a serious mental illness remained untreated . it is easy to understand that a serious condition needs treatment to avoid complications such as suicide , need of inpatient care , and disability . however , studies have shown that the risk of such complications did not differ significantly between mild forms of mental illness compared to moderate forms . in several studies , around half of those affected by psychological distress or psychiatric diagnoses however , even if they seek , the detection rate of psychiatric symptoms is low . a recent meta - analysis of studies regarding general practitioners ability to recognize mild depression showed a detection sensitivity of 56.5% . this emphasizes the importance of further increasing the awareness of mild cases of mental illness . in primary care , a vast majority of patients affected by depression and anxiety present with somatic symptoms [ 11 , 12 ] . somatic complaints include changes in appetite and libido , lack of energy , sleep disturbances , dizziness , palpitations , dyspnoea , and general aches , and pains such as headache , back and other musculoskeletal pain , and gastrointestinal disturbances . identifying persons affected by mental illness , but seeking care for somatic symptoms , is a major difficulty especially in the primary care setting , due to both patient - related issues as well as physician - related issues . it is of importance that somatic symptoms associated with mental health disorders are not confused with somatoform disorders ( i.e. , conversion , somatization , hypochondriasis , and somatization disorder ) . the knowledge of factors influencing care seeking in persons affected by depressive and anxiety disorders in the population is limited . hence , it is important to elucidate factors associated with care seeking in these groups , over all , and factors associated with not seeking care for psychological symptoms . this study aims to describe the prevalence of care seeking among persons with depression and anxiety disorders using data from a population - based study in sweden . first , we aim to study whether affected persons seek care and if care seeking is associated with socioeconomic factors and health status . further , we aim to study if those who seek care for psychological symptoms at the general practitioners differ compared to those who seek care from other health care facilities or do not seek at all . this study is based on the part study ( an acronym in swedish for mental ill - health , work , and relationships ) . part is a longitudinal population - based study of mental health conducted in the stockholm county , sweden . in 1998 - 1999 , 19742 randomly selected swedish citizens aged 2064 years , residing in the stockholm county , were invited to participate and 10441 persons ( response rate 53% ) responded to the self - administrated questionnaire ( baseline ) that included questions on demographic and socioeconomic characteristics , somatic and psychiatric health , and use of drugs . three years after they had answered the first questionnaire ( baseline ) those who answered were reassessed with another similar questionnaire including questions on health care seeking ; 8700 persons participated ( retention rate 83% ) . psychiatrists performed interviews using schedules for clinical assessment in neuropsychiatry ( scan ) , in order to validate the answers of the questionnaires . a comparison between depressions according to the major depression inventory ( mdi ) used in the questionnaire and scan showed good compliance . nonparticipation analysis , using national registers , performed after the first two waves , revealed that the association between gender , age , income , education , country of birth , and psychiatric diagnoses in the national registers was similar among participants and nonparticipants [ 16 , 17 ] . for detailed information about the part study see the technical report . for the purpose of this study we restricted our analyses to the 8387 subjects that participated in both baseline and the first followup , with information on symptoms of depression and anxiety . definitions of anxiety and depressive disorders were made according to dsm - iv criteria , including research criteria , using validated diagnostic scales based on the questionnaire . the included scales were the sheehan patient - rated ( panic ) anxiety scale and the major ( icd-10 ) depression inventory , mdi . social phobia was assessed using the avoidance part of an instrument developed by marks and mathews and for obsessive - compulsive disorders screening questions suggested by the swedish psychiatric association and swedish institute for health services development were used . anxiety disorders included panic syndrome with agoraphobia , agoraphobia without panic syndrome , social phobia , obsessive - compulsive disorder , panic syndrome without agoraphobia , anxiety syndrome due to somatic cause , specific phobia , posttraumatic stress syndrome , general anxiety disorder , and acute stress syndrome . some of the persons affected by major depressive disorder may have a bipolar disorder since there was no scale for manic episodes in the questionnaire . three mutually exclusive groups were created : any depressive disorder ( n = 465 ) , any anxiety disorder ( n = 751 ) , and coexistent depressive and anxiety disorder ( n = 810 ) . in total 2 026 persons ( 24% ) fulfilled the criteria for any depressive or anxiety disorder . health care is accessible to everyone living in sweden , and because of tax subsidies , costs are limited for individuals . both private- and public - funded outpatient clinics are under the same regulations and the patient can choose their preference for the same cost , with exception for those private clinics without affiliation to the public health care system . with regards to psychologists and psychotherapists , there are also private practices without affiliation , and thus not subsidized , a more expensive alternative for the patient . the health care system is organized with a broad base of easy - accessible primary care in health centers , where a variety of health professionals ( doctors , nurses , physiotherapists , psychologists , counsellors , and other staff members ) work . the usual path to seek care is to turn to the health centre to see a specialist in general medicine ( general practitioner , gp ) . the major part of patients is taken care of at this level , but in case the patient needs to see another specialist , he or she is referred by the gp . the gp can also refer the patient to a psychologist or likewise . within the psychiatric sector , it is also possible to directly take contact with an outpatient psychiatric clinic , if it is obvious that the mental problems are severe enough to belong to the psychiatric care . if not , the patient will be redirected to the primary health care centre . the first was have you , due to sleeping problems , personal problems or psychological symptoms , been in contact with one or more of the following during the last 12 months ? psychologist / psychotherapist public or private , general practitioner public or private , other medical / psychological treatment , and alternative medical treatment . seeking care for psychological symptoms was defined as having checked one or more of the response alternatives . the second question was have you , due to bodily symptoms or somatic illness , been in contact with one or more of the following during the last 12 months ? with the following response alternatives : general practitioner public or private , specialist public or private , other medical treatment , and alternative medical treatment . data on country of origin and education was derived from the baseline questionnaire , and all other data was retrieved from the followup . hazardous alcohol use was evaluated using audit ( alcohol use disorders identification test ) [ 26 , 27 ] . education was categorized into three groups : basic compulsory education ( 9 years ) , upper secondary education ( 1012 years ) , and higher education ( college / university , 13 years ) . data from the second wave included household composition , children in household ( permanently or more than half of the time were considered as living with children ) . labour market position included employment / own business , on leave ( studies and parental leave ) , unemployed or in labour market policy measures , disability pension or sick leave for more than a month , and retirement . having a close friend included the answers entirely or fairly true to the question if there was a special person the person felt he / she could get support from . disability last 30 days included those who had been so affected by psychological symptoms / problems that they had not been able at all to pursue the ordinary tasks . somatic illness was measured by a list of 26 somatic disorders , and only those currently treated by a doctor were considered as exposed to somatic illness . the statistical analyses aimed to describe presence of care seeking and possible factors associated with such among persons affected with depression or anxiety disorders . also , the analyses describe what factors could be associated with seeking different types of care . this was done by using cross - tabulation in ibm spss statistics 19.0 on different kinds of care seeking to describe the prevalence of care seeking by demographic , socioeconomic , and psychiatric factors . pearson chi - square tests were used to test for statistical significance . additionally , to analyse differences between persons seeking different kinds of care , one - way analysis and bonferoni tests were used . partially missing answers were treated as missing values in the analyses ( varying from 0.04% on born abroad to at most 2.4% for seeking somatic care ) . a description of the study sample , stratified by depressive and/or anxiety disorders , is presented in table 1 . persons with depression were more often female , young , single , living without children , less often having a close friend and less educated . they reported more often to be on sick leave / disability pension , unemployed , treated for somatic illness , having hazardous alcohol use and were more often affected by disability , compared with those without depression and anxiety . persons with anxiety were more often female , younger and more often had hazardous alcohol use , compared with those without depression and anxiety . persons with comorbid depression and anxiety showed similar differences as those affected by depression and also reported more often having country of origin outside sweden . of those affected by depression and/or anxiety , 47.1% of the persons stated that they had been in contact with some type of health care facility within the last year due to psychological symptoms ; see table 2 . persons who had been seeking help for psychological symptoms were more often female , older , singles , born abroad , or outside the labour market . additionally , they more often had comorbidity factors such as somatic illness , or both depression and anxiety , were more severely affected by depression and more often disabled due to psychological symptoms . when it comes to type of care , 30.4% of the persons affected with depression and/or anxiety had been seeking help for their psychological symptoms at a gp , and 33.5% had been seeking help at other caregivers ; see table 3 . about thirteen percent had reported a gp as their only care provider . in the group that went to the gp , there was an overrepresentation of persons with both depression and anxiety , and disability due to psychological symptoms as well as somatic illness compared to those that did not seek care . this was applicable for both those who had the gp as their only provider , as well as those who also had seen a psychiatrist , psychologist , or other ( alternative / other medical or psychological ) . seeking care to a greater extent to psychiatrists or psychologists was also the case for those with comorbid anxiety and depression , and disability due to psychological symptoms . when it came to those with hazardous alcohol use , there was an overrepresentation among those who had seen both a gp and a psychiatrist / psychologist , compared to the persons that had only attended gp or had not been seeking at all . those that had been seeking gp were also older and more often they had less education , than those who were more likely to not seek care at all , or to seek only a psychiatrist or psychologist . at the gps , persons outside the labour market , on sick leave or disability pension were overrepresented , as well as persons born in another country , both among those who had the gp as their only care provider or in combinations . persons born abroad were also less likely to only have a psychiatrist or psychologist as their only provider . regarding the group that turn only to alternative care or other medical / psychological treatment , there seemed to be no differences among groups except for persons born abroad and less educated persons that were underrepresented . in the present study we found that 52.5% of those affected by depression and/or anxiety disorders did not seek care for psychological symptoms . among those not seeking care for psychological symptoms , two - thirds one reason for seeking for somatic symptoms might be that they primarily have identified the somatic symptoms that often accompanies depression and anxiety , which has been reported in several previous studies [ 12 , 29 , 30 ] . comparison with other studies is somewhat difficult due to different measures on both mental health and of outcomes such as care seeking or treatment . in our study , the proportion seeking care was 47.1% . several other studies have showed prevalence for seeking care for psychological distress or a variety of psychiatric diagnoses ( such as depression , dysthymia , gad , panic disorder , phobias ) , ranging from 36 to 60% [ 39 ] . this shows that the problem with people in need who does not seek help is widely spread . in the present study , persons less likely to seek help were male , younger , born in sweden , living with a partner , employed / on leave for studies or parental leave , retired or had higher education . several studies have reported that prejudices in the general population against male persons affected by mental disorders are higher than against affected females . having a job , being a student , or on parental leave might imply less daytime available in to be spent seeking care . when it comes to labour market position , being on sick leave might be a promoting factor but also a result of care seeking per se and an indicator of severity . summarizing , in the group of persons less likely to seek there is an overrepresentation of individuals that perhaps are less likely to be suspected of being affected of mental problems due to lower load of risk factors as well as assumed to be well adjusted in society . those with milder symptoms and less disability due to psychological symptoms were also less likely to seek . evidently this could be due to less need of care , and it could be argued that minor depression and distress could be resolved without professional help [ 32 , 33 ] . however , mild disorders are increasingly considered clinically significant and detecting them in an early stage might prevent them from turning into serious cases in the future [ 2 , 35 , 36 ] . having been seeking care both at the gps and at the psychiatrists or psychologist / psychotherapists could mean , with regards to how the health care system is organized in sweden , that the gp has referred the patient . this is especially the case when it comes to persons with a hazardous alcohol use that more often had been seeing a gp and a psychiatrist / psychologist . in this study we lacked information on type of clinical specialization but it is likely that it was referrals from gps to clinics specializing in alcohol dependence . there was no gender difference for the category that had seen a gp and a psychiatrist / psychologist / therapist . this could possibly stand for that there is no gender difference when it comes to proportional referrals from the gp , which is gratifying . persons with higher education were less likely to seek care at all , and if they did , they were more likely to turn to a psychiatrist / psychologist . this could possibly stand for a perceived need for a more specific treatment , higher ability to interpret their symptoms as psychological , or more knowledge on possible places to go . that persons under the age of 35 years show the same pattern could maybe stand for partly the same , as in a perceived need for a more specific treatment , but perhaps also for less stigmata surrounding mental problems . the opposite is shown for persons born abroad ; characteristics of migrants ' pathways to psychiatric care have been reported to be delays in seeking professional help , a lower probability of medical referral , frequent involvement of the police and emergency services , and high proportions of compulsory and secure - unit admissions . persons on sick leave , with a disability pension , or unemployed were more likely to see a gp , alone or in combination with psychiatrist / psychologist or other and more likely to seek care . it could be argued that having a long - term psychological health problem might be preceding poorer social functioning resulting in unemployment or sick leave / disability pension . also , contact with a gp or a psychiatrist is a necessity for the medical certificates needed for the social insurance system initiating a sick leave or disability pension , which in part could explain the overrepresentation among these groups . but also , it could stand for a more severe psychological health status or a greater need of treatment . studies have shown that unemployment , as well as sick leave or disability pension per se , can have a negative effect on psychological health . an important factor for not seeking care for psychological symptoms seems to be not having any treatment for somatic illness . this could be an important finding ; if a person has treatment for any somatic problems , he or she already established a relationship to the physician or care - giving facility , which might make bringing up psychological problems easier . older people might also have an easier access to care due to a prior relationship with their gp based on somatic illness or plainly longer experience of care seeking . the category turning only to alternative care seemed to have less to do with the mental illness per se , not varying with severity of illness or disability , but instead with socioeconomic factors that could be argued possibly related to limitations such as high cost or less knowledge of such . in the present population - based study , validated diagnostic scales for assessing anxiety and depression were used [ 4042 ] . one limitation is the cross - sectional design , which limits the possibilities to draw causal conclusions . the self - reported care seeking was measured retrospectively one year back from filling in the questionnaire . the scales measuring symptoms of depression cover the last 14 days and for anxiety the last 30 days , respectively . it could therefore be argued that persons might have symptoms but not yet contacted health care or that persons might fall out of the depression and/or anxiety group population because they have had symptoms previously but not during the last month . however , when examining reports of the duration of symptoms , we found that , of those having any form of depression , one - third had it more than two years , one - third since more than six months , and one - third between two weeks and six months . among those with anxiety , as a general practitioner , it is of great importance to further increase awareness of mild cases of mental illness , especially among groups that might be less expected to be affected by mental illness . despite global reductions in fertility rates , parenthood remains a central life goal in most societies . in recent years , progress in medical technology has offered hope to many infertile couples , especially in the developed world . the us is one of the countries with the highest rate of multiple births in the world . this rate is directly attributed to the increased use of assisted reproductive technologies ( art ) in achieving pregnancy . the number of babies born to gestational surrogates increased to 89% in just 4 years , from 2004 to 2008 . iranian culture generally considers children as divine gifts , and producing children is the fundamental reason for marriage among many couples . having children is generally regarded as strengthening the institution of the family and as a sign of commitment to iranian cultural values . infertility is a social onus for women who are expected to produce children early within marriage in iran . according to the world health organization , over 80 million people ( about 10 - 15% ) of the families experience infertility . in iran , this practice of transferring an embryo or fetus from one womb to another is not forbidden in shiite jurisprudence . most researches have focused on a number of specific issues such as attachment and disclosure to surrogate offspring ; experiences , characteristics , and motivations of surrogate mothers ; and changes in profiles of the commissioning mothers . while some researchers have shown that clients experienced powerful emotions as a result of infertility and its treatment , and women experienced more stress than their partners , and this factor may delay their adaptation with the parenting process , in a retrospective qualitative study in uk , mccallum et al . showed that throughout the pregnancy period of a surrogate mother , couples recalled their levels of anxiety as low and their experience as positive , and in pashmi 's quantitative study , most of the surrogate and commissioning mothers did not consider surrogacy a problematic issue . klock and greenfeld found that in in vitro fertilization ( ivf ) , women experienced more satisfaction with being able to get pregnant than the women who conceived naturally and reported decreased anxiety as the pregnancy progressed . although surrogacy is essentially done with ivf technique , the key difference between surrogacy with other methods of ivf is that commissioning mothers in surrogacy will not experience the physical changes of pregnancy . most of the studies in iran have focused on defining surrogacy , demonstrating the legal aspects , ethical aspects , survey of cultural attitudes about the use of this technique , and ethnography of volunteer surrogate women . but studies about the third - party reproduction in iran are few , and the impacts of these methods on recipients , donors , and children are unknown . we found only one quantitative study , conducted by pashmi et al . in iran , on commissioning mothers experiences . evaluated the experiences of the 30 women involved in surrogacy ( 15 surrogate mothers and 15 commissioning mothers ) and compared them with 30 normal mothers ( women who conceived spontaneously ) in isfahan , a province of iran , using a questionnaire and structured interviews . results showed that the commissioning mothers had happy experiences as well as anxious feelings during the pregnancy of the surrogate mother . they were anxious about the health of both the surrogate mother and the baby . based on mercer , identifying a mother 's unique concerns and available resources to address these concerns and reinforcing her caretaking skills are important in fostering the mother 's sense of competence as she works on gaining a maternal identity . nurses are in a unique position to have long - term positive effects on mothers during this transition , and have a leadership role in providing anticipatory guidance and education that is grounded in the unique experiences of primigravid and multigravid women . there are a few studies that investigated the quality of commissioning mothers experiences such as their anxiety and stress . since the number of spouses treated by art such as surrogacy is growing worldwide , it is crucial to understand their emotional and psychological reactions toward surrogacy during the 9 months of the waiting period . this is the first qualitative study in iran , which assessed the experiences of commissioning mothers . with their diverse culture and social contexts , developing countries demand much more context - dependent studies this study has aimed to explore and explain the nature of concerns ( experiences ) of commissioning mothers . the aim of this study was to explore and explain the nature of concerns ( experiences ) of commissioning mothers . the aim of this study was to explore and explain the nature of concerns ( experiences ) of commissioning mothers . content analysis is a research method that has come into wide use in health studies in recent years . this type of design is usually appropriate when an existing theory or research literature on a phenomenon is limited . royan research centre consists of six departments ( endocrinology and female infertility , epidemiology and reproductive health , reproductive genetics , reproductive imaging , andrology , and embryology ) and one clinic actively working on different aspects of infertility and the development of new methods for infertility treatment . our participants were first - time commissioning mothers , during the waiting period ( pregnancy of the surrogate mother ) or after that ( child rearing period ) . they had to be able to speak persian and willing to participate in the research . participants were selected from gestational surrogacy cases ( the conceptions were with own gametes ) , as gestational surrogacy is more prevalent in iran than partial surrogacy and we had a better access to them . women who were in a waiting list for finding a surrogate by the infertility centers or those who were waiting to know the result of embryo implantation and the women whose embryo was aborted after implantation were not included in this study . it means for selection , at first , we clarified mothers who had the first - time commissioning mothers , during the waiting period ( pregnancy of the surrogate mother ) or after that ( child rearing period ) , and then , interviews were conducted . the first author ( mz ) conducted all the interviews from november 2010 to august 2011 . of the 10 commissioning mothers suggested by royan research centre , 7 agreed to participate in the study and the other 3 mothers refused due to a fear of disclosure . finally , this research was conducted with 24 interviews , and 16 people including 12 commissioning mothers , 2 surrogate mothers , and 2 of the infertility center staffs who directly and continuously dealt with the mothers ( social workers ) . the interviews were continued to obtain richer data and stopped when no new information was obtained . the analysis starts with reading all data repeatedly to achieve immersion and obtain a sense of the whole . then , data are read word by word to derive the codes by first highlighting the exact words from the text that appear to capture key thoughts or concepts . as this process continues , these labels often come directly from the text and then become the initial coding scheme . then , the codes are sorted into categories based on how different codes are related and linked . coding and analysis was done by a single researcher who was the first author ( mz ) , and the other researcher checked them again and confirmed them as it is necessary for credibility . the interviews were recorded and typed ( in persian ) as soon as possible in the exact words , along with all nonverbal gestures such as crying , laughter , or silence . then , they were entered in maxqda2007 software that makes it easier to organize and keep an overview over memos and contents . they were read and reviewed several times and , eventually , disintegrated into meaning units . after reviewing each meaning unit , next , the primary open codes were classified based on conceptual and meaning similarities and put in the main categories which were more general and more conceptual . the categories were finally reduced to the two main themes of cultural dilemma and uncertain waiting . in order to maximize credibility of the findings in this study , prolonged engagement with data ( 1 year ) , members check , externals check , and triangulation recourses ( commissioning mothers , surrogates , and infertility center staffs ) were used . in order to keep confidentiality , as some of the commissioning mothers did not have any opportunity to express their emotions and concerns , they found the interviews so relaxing since they could release their pressures and this matter increased the credibility of the study . the study process was carefully recorded step by step so that conformability , validity , transferability , and audit of the study were achieved and another researcher could follow this research . before data collection , an approval was obtained from the ethics committee of tarbiat modarres university and royan institute . by a phone call in the beginning , the research purpose , interviewing techniques , confidentiality of information , and the right to withdraw from the study were explained to the participants . a suitable time and place for an interview interviews were conducted individually in a quiet environment like home , the park near participants homes , or at royan research centre . royan research centre consists of six departments ( endocrinology and female infertility , epidemiology and reproductive health , reproductive genetics , reproductive imaging , andrology , and embryology ) and one clinic actively working on different aspects of infertility and the development of new methods for infertility treatment . our participants were first - time commissioning mothers , during the waiting period ( pregnancy of the surrogate mother ) or after that ( child rearing period ) . they had to be able to speak persian and willing to participate in the research . participants were selected from gestational surrogacy cases ( the conceptions were with own gametes ) , as gestational surrogacy is more prevalent in iran than partial surrogacy and we had a better access to them . women who were in a waiting list for finding a surrogate by the infertility centers or those who were waiting to know the result of embryo implantation and the women whose embryo was aborted after implantation were not included in this study . it means for selection , at first , we clarified mothers who had the first - time commissioning mothers , during the waiting period ( pregnancy of the surrogate mother ) or after that ( child rearing period ) , and then , interviews were conducted . the first author ( mz ) conducted all the interviews from november 2010 to august 2011 . of the 10 commissioning mothers suggested by royan research centre , 7 agreed to participate in the study and the other 3 mothers refused due to a fear of disclosure . from the infertility centers of other provinces , finally , this research was conducted with 24 interviews , and 16 people including 12 commissioning mothers , 2 surrogate mothers , and 2 of the infertility center staffs who directly and continuously dealt with the mothers ( social workers ) . the interviews were continued to obtain richer data and stopped when no new information was obtained . the analysis starts with reading all data repeatedly to achieve immersion and obtain a sense of the whole . then , data are read word by word to derive the codes by first highlighting the exact words from the text that appear to capture key thoughts or concepts . as this process continues , these labels often come directly from the text and then become the initial coding scheme . then , the codes are sorted into categories based on how different codes are related and linked . coding and analysis was done by a single researcher who was the first author ( mz ) , and the other researcher checked them again and confirmed them as it is necessary for credibility . the interviews were recorded and typed ( in persian ) as soon as possible in the exact words , along with all nonverbal gestures such as crying , laughter , or silence . then , they were entered in maxqda2007 software that makes it easier to organize and keep an overview over memos and contents . they were read and reviewed several times and , eventually , disintegrated into meaning units . after reviewing each meaning unit , next , the primary open codes were classified based on conceptual and meaning similarities and put in the main categories which were more general and more conceptual . the categories were finally reduced to the two main themes of cultural dilemma and uncertain waiting . in order to maximize credibility of the findings in this study , prolonged engagement with data ( 1 year ) , members check , externals check , and triangulation recourses ( commissioning mothers , surrogates , and infertility center staffs ) were used . in order to keep confidentiality , as some of the commissioning mothers did not have any opportunity to express their emotions and concerns , they found the interviews so relaxing since they could release their pressures and this matter increased the credibility of the study . the study process was carefully recorded step by step so that conformability , validity , transferability , and audit of the study were achieved and another researcher could follow this research . before data collection , an approval was obtained from the ethics committee of tarbiat modarres university and royan institute . by a phone call in the beginning , the research purpose , interviewing techniques , confidentiality of information , and the right to withdraw from the study were explained to the participants . a suitable time and place for an interview interviews were conducted individually in a quiet environment like home , the park near participants homes , or at royan research centre . the demographic characteristics of commissioning mothers and surrogate mothers are shown in tables 2 and 3 , respectively . demographic characteristics of mothers demographic characteristics of surrogates the experiences of the participants were classified in two main themes : cultural dilemma with three subthemes : social taboo , concerns about disclosure to others and child , concerns about altered maternal and child 's identityuncertain waiting with three subthemes : concerns about health of fetus and surrogate , concerns about unknown surrogate , and concerns about lack of preparation for maternal role [ table 4 ] . cultural dilemma with three subthemes : social taboo , concerns about disclosure to others and child , concerns about altered maternal and child 's identity uncertain waiting with three subthemes : concerns about health of fetus and surrogate , concerns about unknown surrogate , and concerns about lack of preparation for maternal role [ table 4 ] . social taboos , 2 . concerns about disclosure to others and child , and 3 . infertile couples decide to use this technique after desperate years of trying other treatment modalities and prolonged resistance toward using surrogacy , but concerns about other people 's blaming points of view about surrogacy turn into a frightening experience and a taboo . one of these mothers stated her experience as follows : social taboos have rooted in the cultural barriers of surrogacy usage in the society . infertile couples decide to use this technique after desperate years of trying other treatment modalities and prolonged resistance toward using surrogacy , but concerns about other people 's blaming points of view about surrogacy turn into a frightening experience and a taboo . one of these mothers stated her experience as follows : well , for the things people say . my in - laws for instance , my mother in - law , for example , was saying that one of our relatives had an implantation ! although she is an educated person , supposedly , they should know better , but still their reaction was so bad . these all were too much for me to take , too much to take . ( m7 ) 2.mothers were also worried about their secret disclosure to others and child . concern about revelation of using surrogacy to other was reflected in one of the statements : mothers were also worried about their secret disclosure to others and child . concern about revelation of using surrogacy to other was reflected in one of the statements : i mean i was anxious all t he time about this idea what should i do if somebody finds out? ( m1 ) all mothers also were worried about disclosure to their child and its revelation by relatives or young children . they believed this can not be done suddenly without preparation and should be planned to be revealed by parents themselves . they were afraid of the child to be informed when he / she does not have proper understanding of the events yet and the ability to analyze the truth , so that disclosure by others may lead to huge psychological trauma and his / her blaming parents as liars . one of the mothers said : i feel that telling this subject to the child has to be done at a certain age . i mean , we should reveal the truth when she is mature enough to be able to understand what you are telling her . if she is told all of a sudden , she would think of me as a liar because i did nt tell her the truth , she would not believe me and i might struggle with her a lot . ( m2 ) 3.another concern in this category was altering the maternal and child 's identity . mothers were worried about their children not being accepted by family , relatives , and peers , and that they might look differently to the child and have social stigma . these mothers even suffer more after the child 's birth . mothers were worried about their children not being accepted by family , relatives , and peers , and that they might look differently to the child and have social stigma . what if someone does nt respect my child , treat him like a foster kid , what should i do ? ( m6 ) statements of social workers in the infertility center confirmed this kind of emotions in mothers : they are worried about the relationship between their relatives and their newborn babies . concerns about altering maternal identity means that they were worried about not being accepted as a real mother by others and even by the baby . mothers were worried that in case of a disclosure , they may not be accepted as the genetic mother . i do nt know whether they will accept me as a mother now if they know this is my baby . i have this kind of baby.(m6 ) they were also concerned about not being assumed as a real mother by the child , for example : i am afraid that in future my baby would tell me that we found him in the street and adopted him or got him from a foster home . 1.mothers were strongly concerned about the fetus 's health and the possibility of abortion , stillbirth or disabilities , and premature labor . these statements were repeated by almost all mothers and are some of the disturbing factors in mothers psychological well - being . mothers were strongly concerned about the fetus 's health and the possibility of abortion , stillbirth or disabilities , and premature labor . these statements were repeated by almost all mothers and are some of the disturbing factors in mothers psychological well - being . well , one thing that disturbs me is that i sometimes tell myself maybe because the baby is injected until 9 months long , i have heard many times about premature babies birth in the 7 or 8 months , or miscarriage , or the baby born with deformities . even before birth in the 9th month ( m4 in waiting period ) based on most of the mothers thoughts , it was clear that they had always been worried to hear horrible news about their fetus . i thought all the time something would happen to them ( her three fetuses ) . what would happen now ? really , every night , i have this nightmare that the surrogate mother is sending me an sms . ( m9 ) mothers concerns were increased in critical conditions such as a threatened abortion or decreasing fluid of amniotic sac partially during delivery . during the last month after check - up , the dr . told me that i was dehydrated , my amniotic fluid was too little , and the mother was so horrified . ( sm2 ) since the physical and psychological relaxations of pregnant women directly affect the baby 's physical and psychological well - being , mothers were worried about the surrogates comfort : when i call her up , the first thing i ask her is how she is doing . and then , i ask about the baby . for example , i ask her , is everything ok in your home ? are you relaxed? ( m4 in waiting period ) in addition to the effect of surrogate 's health on fetus , mothers had also had an altruistic feeling to the surrogate and they were worried about the possibility of a physical damage or even death because of their child 's delivery . ( m2 ) 2.another important subtheme of uncertain waiting was the concerns about an unfamiliar surrogate . in the beginning of the process , mothers did not have enough knowledge about the surrogate ; they were worried about giving their fetus to a strange family . they were also terrified that the surrogate might not adhere to the religious principles during pregnancy or might not give the newborn after delivery or may extort the couples after child 's birth continuously or have intentional abortion , which are all related to their lack of knowledge about the surrogate leading to unreliability , for example : another important subtheme of uncertain waiting was the concerns about an unfamiliar surrogate . in the beginning of the process , mothers did not have enough knowledge about the surrogate ; they were worried about giving their fetus to a strange family . they were also terrified that the surrogate might not adhere to the religious principles during pregnancy or might not give the newborn after delivery or may extort the couples after child 's birth continuously or have intentional abortion , which are all related to their lack of knowledge about the surrogate leading to unreliability , for example : i told my husband , do nt take me home tonight. he asked me how can i leave my baby in her womb and go home? i just could nt . ( m3 ) 3.the other subtheme of uncertain waiting was about the lack of preparation to becoming a mother for different reasons . although most of these women became a mother at an older age and they had previous experience of taking caring of their relatives children , they were more concerned about the inability in taking care of their own child , insomnia , and inability for emotional attachment with the child , and so on ; for example : the other subtheme of uncertain waiting was about the lack of preparation to becoming a mother for different reasons . although most of these women became a mother at an older age and they had previous experience of taking caring of their relatives children , they were more concerned about the inability in taking care of their own child , insomnia , and inability for emotional attachment with the child , and so on ; for example : during these 9 months , you wish to have what you have always wanted in one thing , the baby . now , when you got what you wanted for so long , you found out you are not ready . my god when this child is born , i am not even able to touch him . how can i make a relationship with him? i think those 9 months of pregnancy makes you prepared for that . social taboos , 2 . concerns about disclosure to others and child , and 3 . infertile couples decide to use this technique after desperate years of trying other treatment modalities and prolonged resistance toward using surrogacy , but concerns about other people 's blaming points of view about surrogacy turn into a frightening experience and a taboo . one of these mothers stated her experience as follows : social taboos have rooted in the cultural barriers of surrogacy usage in the society . infertile couples decide to use this technique after desperate years of trying other treatment modalities and prolonged resistance toward using surrogacy , but concerns about other people 's blaming points of view about surrogacy turn into a frightening experience and a taboo . one of these mothers stated her experience as follows : well , for the things people say . my in - laws for instance , my mother in - law , for example , was saying that one of our relatives had an implantation ! although she is an educated person , supposedly , they should know better , but still their reaction was so bad . these all were too much for me to take , too much to take . ( m7 ) 2.mothers were also worried about their secret disclosure to others and child . concern about revelation of using surrogacy to other was reflected in one of the statements : mothers were also worried about their secret disclosure to others and child . concern about revelation of using surrogacy to other was reflected in one of the statements : i mean i was anxious all t he time about this idea what should i do if somebody finds out? ( m1 ) all mothers also were worried about disclosure to their child and its revelation by relatives or young children . they believed this can not be done suddenly without preparation and should be planned to be revealed by parents themselves . they were afraid of the child to be informed when he / she does not have proper understanding of the events yet and the ability to analyze the truth , so that disclosure by others may lead to huge psychological trauma and his / her blaming parents as liars . one of the mothers said : i feel that telling this subject to the child has to be done at a certain age . i mean , we should reveal the truth when she is mature enough to be able to understand what you are telling her . if she is told all of a sudden , she would think of me as a liar because i did nt tell her the truth , she would not believe me and i might struggle with her a lot . ( m2 ) 3.another concern in this category was altering the maternal and child 's identity . mothers were worried about their children not being accepted by family , relatives , and peers , and that they might look differently to the child and have social stigma . mothers were worried about their children not being accepted by family , relatives , and peers , and that they might look differently to the child and have social stigma . these mothers even suffer more after the child 's birth . what if someone does nt respect my child , treat him like a foster kid , what should i do ? ( m6 ) statements of social workers in the infertility center confirmed this kind of emotions in mothers : they are worried about the relationship between their relatives and their newborn babies . concerns about altering maternal identity means that they were worried about not being accepted as a real mother by others and even by the baby . mothers were worried that in case of a disclosure , they may not be accepted as the genetic mother . i do nt know whether they will accept me as a mother now if they know this is my baby . i have this kind of baby.(m6 ) they were also concerned about not being assumed as a real mother by the child , for example : i am afraid that in future my baby would tell me that we found him in the street and adopted him or got him from a foster home . concerns about health of the fetus and surrogate , 2 . unknown surrogate , and 3 . 1.mothers were strongly concerned about the fetus 's health and the possibility of abortion , stillbirth or disabilities , and premature labor . these statements were repeated by almost all mothers and are some of the disturbing factors in mothers psychological well - being . mothers were strongly concerned about the fetus 's health and the possibility of abortion , stillbirth or disabilities , and premature labor . these statements were repeated by almost all mothers and are some of the disturbing factors in mothers psychological well - being . well , one thing that disturbs me is that i sometimes tell myself maybe because the baby is injected until 9 months long , i have heard many times about premature babies birth in the 7 or 8 months , or miscarriage , or the baby born with deformities . even before birth in the 9th month ( m4 in waiting period ) based on most of the mothers thoughts , it was clear that they had always been worried to hear horrible news about their fetus . i thought all the time something would happen to them ( her three fetuses ) . really , every night , i have this nightmare that the surrogate mother is sending me an sms . ( m9 ) mothers concerns were increased in critical conditions such as a threatened abortion or decreasing fluid of amniotic sac partially during delivery . during the last month after check - up , the dr . told me that i was dehydrated , my amniotic fluid was too little , and the mother was so horrified . ( sm2 ) since the physical and psychological relaxations of pregnant women directly affect the baby 's physical and psychological well - being , mothers were worried about the surrogates comfort : when i call her up , the first thing i ask her is how she is doing . and then , i ask about the baby . for example , i ask her , is everything ok in your home ? are you relaxed? ( m4 in waiting period ) in addition to the effect of surrogate 's health on fetus , mothers had also had an altruistic feeling to the surrogate and they were worried about the possibility of a physical damage or even death because of their child 's delivery . ( m2 ) 2.another important subtheme of uncertain waiting was the concerns about an unfamiliar surrogate . in the beginning of the process , mothers did not have enough knowledge about the surrogate ; they were worried about giving their fetus to a strange family . they were also terrified that the surrogate might not adhere to the religious principles during pregnancy or might not give the newborn after delivery or may extort the couples after child 's birth continuously or have intentional abortion , which are all related to their lack of knowledge about the surrogate leading to unreliability , for example : another important subtheme of uncertain waiting was the concerns about an unfamiliar surrogate . in the beginning of the process , mothers did not have enough knowledge about the surrogate ; they were worried about giving their fetus to a strange family . they were also terrified that the surrogate might not adhere to the religious principles during pregnancy or might not give the newborn after delivery or may extort the couples after child 's birth continuously or have intentional abortion , which are all related to their lack of knowledge about the surrogate leading to unreliability , for example : i told my husband , do nt take me home tonight. he asked me how can i leave my baby in her womb and go home? i just could nt . ( m3 ) 3.the other subtheme of uncertain waiting was about the lack of preparation to becoming a mother for different reasons . although most of these women became a mother at an older age and they had previous experience of taking caring of their relatives children , they were more concerned about the inability in taking care of their own child , insomnia , and inability for emotional attachment with the child , and so on ; for example : the other subtheme of uncertain waiting was about the lack of preparation to becoming a mother for different reasons . although most of these women became a mother at an older age and they had previous experience of taking caring of their relatives children , they were more concerned about the inability in taking care of their own child , insomnia , and inability for emotional attachment with the child , and so on ; for example : during these 9 months , you wish to have what you have always wanted in one thing , the baby . now , when you got what you wanted for so long , you found out you are not ready . i kept telling myself , my god when this child is born , i am not even able to touch him . how can i make a relationship with him? i think those 9 months of pregnancy makes you prepared for that . mothers participating in this study had chosen surrogacy as their last hope , after years of infertility and sometimes after repeated unsuccessful treatments , and this turns them to sensitive people as the stress during the waiting period can intensify the remaining effects of infertility in these individuals . study findings have shown semantic elements of commissioning mothers concerns ( experiences ) in the two themes of cultural dilemma and uncertain waiting . these themes suggested that the experience of becoming a mother through surrogacy is highly stressful for mothers , and during the waiting period , they experience such a stressful situation in which according to one of the mothers , 9 months of waiting period passes like nine centuries . the study findings have shown that the traditional society of iran has not universally accepted surrogacy and cultural rejection is a great stressful element for commissioning families . in pashmi 's study in iran , most of the concerns were related to the social attitude toward surrogacy . abbasi - shvazy et al . showed that women who receive donor oocytes believe that the iranian community has not accepted art yet , as in the case of a disclosure , infertility stigma , which they have been attributed to , is transferred to their children as an illegitimate baby . mothers in the above - mentioned study also expressed their fear of disclosure of surrogacy to their children , and that their children might feel being a different human being in comparison with their friends . in hershberger . 's phenomenological study on received donor oocytes in california , women addressed social acceptance of this method as a social support . they expressed their concern as disclosing the nature of conception may have the potentiality to cause discordances in the relationships and identities within the family , maternal identity , and maternal insecurity . fear of social stigma and rejection by the society were the other concerns of these women . the predominant traditional cultural and social context and lack of cultural preparation were the major effective factors concerning mothers emotions , which , based on our findings , led to the concerns about the others reactions and altered maternal identity . it should also be considered that full surrogacy is a completely different condition from oocyte donation in which mothers do not experience pregnancy , which is important in collation of findings of different studies . also , in kirkman 's study , women who became mothers through egg donation mentioned concerns about their maternal identity . in contrast with the present study , the retrospective study of mccallum et al . in uk showed a low level of anxiety in couples throughout the pregnancy of a surrogate mother . in their study , the majority of the couples families reacted either positively or neutrally to the news , with only 7% reporting any negative reactions from the family . also , in contrast to our findings , studies in uk showed that families using surrogacy ( complete and partial ) acted openly in exposing the truth to the child . it seems that the main reason for this difference is different cultural contexts in iran and uk . iran is in a historical transition phase from tradition to modernism , which simultaneously causes special conflicts in utilization of new treatment modalities such as surrogacy by families . so , giving the public sufficient information about surrogacy and the other supplemental therapies for infertility by media such as tv and newspapers can improve the negative attitude toward surrogacy and assist most of the surrogate mothers and infertile couples . concerns about fetus 's and surrogate 's health , lack of preparation for motherhood role , and an unfamiliar surrogate were all aspects of uncertain waiting , so that the mothers imagined an uncertain future for them during this period . being anxious about the health of both the surrogate mother and van den akker reported fear of the treatment failure and a normal baby worries in 15% and 8% of the mothers , respectively . it is clear that even in natural pregnancies , mothers are concerned about the child 's health , the consequences of the waiting period , and lack of their preparation for a maternal role , but based on commissioning mothers , their unawareness about fetus was an important factor that intensified their concerns . reported a high level of anxiety about pregnancy security and fetal health among the participants , which is similar to our findings . uncertainty about the outcome had a great impact on the stress level of the women experiencing ivf , which is consistent with the study of ardent et al . all findings about these mothers must be interpreted cautiously , as this method is different from other techniques of mothering , with no waiting period . also , in another study comparing the women with a natural pregnancy with ivf , the results showed that both ivf mothers and ivf partners were more anxious about losing the pregnancy compared to couples who conceived naturally , possibly as they themselves had a difficulty in getting pregnant successfully . ivf partners had a high degree of anxiety about the expected baby concerning not being healthy or normal , but their anxiety decreased as the pregnancy progressed to similar levels of those who conceived naturally . in the present study , only one mother reported that her anxiety decreased as the pregnancy progressed in the surrogate mother . meanwhile , other participants felt more anxious in the later stages of the waiting period . this difference may be related to perceiving the movements of fetus through pregnancy progression of the mothers with other infertility treatments , which makes mothers more relaxed and confident about their fetus 's health , while this can not be felt by surrogacy commissioning mothers , so they become more concerned about losing the baby and missing their dreams of becoming a mother while waiting . van den akker also found that commissioning mothers anxiety ( in full or partial surrogacy ) could be significantly intensified in the final stages of surrogate pregnancy . lack of knowledge about the surrogate , relying on the surrogate mother , and fear of not delivering the baby after birth were the other stated concerns of those mothers , which were also partially found in van den akker 's study . in her study , 23% of commissioning mothers were worried that relinquishing the baby may become emotionally difficult for the surrogate mother . other types of worries included emotional difficulties for commissioning ( 3% ) or surrogate mother ( 19% ) . keen ( as cited in teman ) stated that 99% of the surrogate mothers willingly relinquished the child as they had contractually agreed to do . less than 0.1% of surrogacy cases end up in court battles . based on mccallum et al . 's report , only one of the surrogate mothers had doubts about relinquishing the baby and the others did not have any problem to do that . in the present study and in pashmi 's study in iran , all of the surrogates gave the baby to the commissioning mother immediately after delivery and there was not any problem reported . van den akker believes that the surrogate agency information appears to be quite successful in assisting the surrogates to achieve a cognitively consonant state . in conclusion , the study results found that mothers experienced a continuous stressful waiting period and psychological and social insecurity . this study suggests being more supportive for commissioning mothers through psychological consultations , application of relaxation techniques , and family education classes . in these classes , even though this study tried to collect in - depth information by utilizing various resources on one hand and the participating mothers were studied in both waiting period and after child birth on the other hand , there were some limitations . due to unavailability of partial surrogate samples , this research has been conducted only on gestational / full surrogacy , while partial surrogacy may yield different results . in addition , cultural and , particularly , religious differences in women can influence their experiences and perceptions . the result of this study can be used as a practical guide for nurses and other team members of art in iran and other muslim countries where art is not forbidden . in addition , in the countries with similar culture , where these technologies are not permitted , it can be used in decision making about this matter . provision of the best health care , as the purpose of nursing , requires careful assessment and evaluation of people 's experiences about diseases and treatment methods . in order to provide sophisticated care , art nurses should first know about clients experiences , and then , plan nursing care accordingly . the study results showed that psychological support is necessary before a successful intervention during the waiting period and after child birth . it is recommended that since surrogacy is considered as a high - risk psychological experience , these mothers should be cared for and receive special consultations before making such a decision . these findings help the mothers through nurses interventions in health care clinics or wherever they provide nursing care . nurses who work in art field should identify their roles and abilities to positively affect mothers experiences . increasing the use of art necessitates nurses planning and preparation to improve the health of the families who use this technology and are the fundamental institutions in the society .

background . in primary care , a vast majority of patients affected with depression and anxiety present with somatic symptoms . detection rate of psychiatric symptoms is low , and knowledge of factors influencing care seeking in persons affected by depressive and anxiety disorders on a population level is limited . objective . this study aims to describe if persons , affected by depression and anxiety disorders , seek care and which type of care they seek as well as factors associated with care seeking . method . data derives from a longitudinal population - based study of mental health conducted in the stockholm county in 19982010 and the present study includes 8387 subjects . definitions of anxiety and depressive disorders were made according to dsm - iv criteria , including research criteria , using validated diagnostic scales . 2026 persons ( 24% ) fulfilled the criteria for any depressive or anxiety disorder . results . forty - seven percent of those affected by depression and/or anxiety had been seeking care for psychological symptoms within the last year . a major finding was that seeking care for psychological symptoms was associated with having treatment for somatic problems . conclusions . as a general practitioner , it is of great importance to increase awareness of mild mental illness , especially among groups that might be less expected to be affected . background : there are a few studies about commissioning mothers understanding from the surrogacy during 9 months of waiting for delivery in iran and other countries . this study was conducted with an aim to explore and explain the nature of concerns ( experiences ) of commissioning mothers.materials and methods : a qualitative design with a conventional content analysis approach was used to gather and analyze the experiences of commissioning mothers . they were selected from royan research centre and other infertility centers in iran . after purposive sampling for the selection of the participants , unstructured interviews were held for data collection . twenty - four unstructured interviews were conducted with 12 commissioning mothers , 2 surrogate mothers , and 2 infertility center social workers who directly and continuously dealt with these mothers.results:two main themes emerged from the data analysis : 1 . cultural dilemma ( consisting of three subthemes : social taboo , concerns about disclosure to others and the child , concerns about altering maternal and child 's identity , and 2 . uncertain waiting ( consisting of three subthemes : concerns about health of fetus and surrogate , concerns about an unfamiliar surrogate , and concerns about lack of preparation for maternal role).conclusions : the study reveals the importance of maternal emotional care in this group and introduces a new arena for nurses activity . these findings help the mothers by nurses activities in health care clinics and anywhere they deliver nursing care .

characterization of the bioactive constituents from the black pigmented ink has resulted in a handful of chemical elucidations . the ink from the molluscs has created a great interest towards its bioactive molecules with promising antibacterial , antitumour , antileukemic , and antiviral activities . the ink is ejected from the ink gland of the squid loligo duvauceli through the ink duct to escape from its predators . the black pigment was found to be melanin and the process of melanogenesis was explained in the ink gland of sepia sp . . the ink is a complex mixture of organelles , premelanosomes , melanosomes , granules , proteic material ( enzymes ) , glucosamine , and phospholipids in suspension . at the moment of extraction the ink gland has been also shown to contain a variety of melanogenic enzymes as tyrosinase , dopachrome tautomerase , and peroxidase . the ink has also various primary roles in the world of alternative medicine and has the widest range of therapeutic application . despite these reports there is no considerable interest shown towards the purification procedures of the l. duvauceli 's ink . potential chemical cues in squid ink have been identified and quantified using reverse - phase , high - performance , liquid chromatography ( rp - hplc ) . so this study is aimed at exploring the active bioconstituents of the ink by silica gel column chromatography and the gas chromatography - mass spectroscopic analysis ( gc - ms ) for its antimicrobial constituents . the collection of ink and the crude solvent extraction of the constituents from l. duvauceli ink was done by the method followed earlier . the crude extracts were subjected to sterility checking after exposing the extracts under uv light for 2 hrs . 5 mg of each extract was mixed in sterile nutrient broth and was incubated for 2 hrs which was plated onto nutrient agar for checking the sterility of the extracts . the antimicrobial activity of the crude extracts was performed by conventional agar well diffusion method . the n - hexane extract has scored in our earlier reports a high antimicrobial property against the clinical bacterial and fungal isolates . thus the n - hexane extract was chosen for the further fractionation by silica gel column chromatography . separation of the active biomolecules from the crude n - hexane extract was done by silica gel column chromatography . briefly , 10 gm of the crude n - hexane extract was subjected for fractionation using silica gel column . the crude extract was adsorbed on to silica gel ( 100200 mesh , sisco ) and chromatographed employing a step gradient solvent system from low to high polarity . the starting solvent system was 100% n - hexane and subsequently the polarity was increased by varying the solvent concentration with ethyl acetate ( etoac ) . the solvent gradient for the chromatogram is 100% hexane , 20 : 80 etoac / hex , 40 : 60 etoac / hex , 60 : 40 etoac / hex , 80 : 20 etoac / hex , 100% etoac , 100% diethyl ether , 20 : 80 ethanol / etoac , 40 : 60 ethanol / etoac , 60 : 40 ethanol / etoac , 80 : 20 ethanol / etoac , 100% ethanol , 20 : 80 water / etoac , 50 : 50 water / etoac , and 100% water . in order to select the best mobile phase for eluting the fractions , 5 l of each eluted fraction the fractions that showed the elution of similar compounds were pooled and concentrated under vacuum below 40c using heidolph , ve-11 rotaevaporator for 30 min . high performance thin layer chromatography ( hptlc ) analysis combined fractions were kept under air current to facilitate drying . the concentrated fraction was obtained and subjected to sterility checking as mentioned earlier . the active fraction was stored at 4c in sterile glass brown bottles until used for the bioactivity studies . the antimicrobial activity of isolated fractions was checked against escherichia coli ( atcc 25922 ) , klebsiella pneumoniae ( atcc 10031 ) , staphylococcus aureus ( atcc 25923 ) , candida albicans ( atcc 10231 ) , and lactobacillus acidophilus [ mtcc 447 ] . briefly , mueller hinton agar plate was divided into two halves and 50 l of inoculum of each test organism was spread as lawn cultures on the same plate to achieve a confluent growth at each half . the agar plates were allowed to dry and wells or cups of 8 mm were made with a sterile agar borer on the inoculated agar plates . 10 mgs of the pooled fraction was mixed with dmso and was made ready for the study . a 50 l volume of the active fraction was propelled directly into the wells of the inoculated specific media agar plates for each test and control organism . erythromycin ( 30 g ) and amphotericin b ( 100 u ) were used as the positive controls for the bacteria and the yeast , respectively . the plates were allowed to stand for 10 minutes for diffusion of the extract to take place and were incubated at 37c for 24 h. after incubation the plates were observed for the zone of inhibition around the wells and the zone of inhibition was measured using an antibiotic sensitivity measuring scale ( himedia , mumbai ) . determination of mic value for the active antimicrobial fraction was determined by microbroth dilution method . serial dilutions of the active fraction were done in a 96-well microtitre plate with dmso . the dilution factor was 5 , 2.5 , 1.25 , 0.625 , 0.312 , and 0.156 mg / ml . to each dilution 100 l of the culture broths of the test organisms was added in their respective wells and the plate was incubated at 37c for 24 hrs . after incubation the spectrophotometric analysis was performed and the od values were recorded . the mbc value was confirmed by microbial spot checker board method where 3 l of each dilution was spotted onto mueller hinton agar plates and incubated at 37c for 24 hrs . after incubation the spot showing the complete absence of microbial growth indicates the minimum bactericidal value and the spot showing the visible decrease in the number of colonies indicates the minimum inhibitory concentration ( mic value ) . the active fraction was then subjected to gc - ms analysis with the split injection ratio of 1 : 20 in hexane in shimadzu gc - ms qp 2010 ( japan ) gas chromatograph with capillary column length of 30 m , 0.25 mm diameter , and 0.25 m of film thickness . gc - ms operating conditions were as follows : injector temperature at 280 , column oven temperature at 45 , flow control mode at linear velocity , and column flow of helium ( 99.9% purity ) at 1.40 ml / min . oven temperature programme was maintained at 45c for 2 min and 300c for 10 min with overall holding time of 36.5 min . mass spectra conditions applied were as follows : electron impact at 40 ev , ion source temperature at 200c , and interface temperature at 240c . the n - hexane extract scoring a high antimicrobial activity upon column fractionation over silica gel yielded a total of 8 fractions . elution with hex / etoac in the ratio of 4 : 1 yielded the active fraction . the active fraction was subjected to tlc , hptlc , and gc - ms analysis . tlc profile showed a single spot with a retention factor of 0.76 ( figure 1 ) . the same plate was subjected to hptlc analysis , with a scanning wavelength of 254 nm . the chromatogram showed a single peak obtained as calibration spectrum data with a noise level at 0.072 mv , camag software , and scanned with scanner ii with area normalization of 83.91% that indicated the maximum extraction with hex / etoac . antimicrobial bioassay revealed that the active fraction possesses a high antibacterial activity against the test organisms ( table 1 ) . the zone size was measured as 18 mm for e. coli and k. pneumoniae , 16 mm for s. aureus , 23 mm for c. albicans , and 18 mm for l. acidophilus ( figure 2 ) . the mbc value was determined as an average of 2.5 mg / ml for e. coli , k. pneumoniae , and c. albicans , 5 mg / ml for s. aureus and l. acidophilus . the microbial spot checker ( figure 3 ) board method yielded complete absence of the growth at the spot inoculated with the determined mbc value . the previous dilution that showed the visible decrease in the number of colonies was determined as the mic and was deduced as 1.25 mg / ml for e. coli , k. pneumoniae , and c. albicans and 2.5 mg / ml for s. aureus and l. acidophilus . the bioactive fraction upon gc - ms analysis revealed a chromatogram showing nine peaks with bis(2-ethylhexyl ) phthalate [ behp ] possessing the highest percentage of area normalisation ( 91% ) ( figure 4 ) . the mass spectrum was found to be superimposable ( > 93 ) with that of the authentic compound from the gc - ms library . based on the gc - ms analysis the active fraction was structurally elucidated as bis(2-ethylhexyl ) phthalate . the chromatogram also showed the presence of other minor compounds such as octadecane ( 0.29% ) , naphthalene ( 0.13% ) , tetradecane ( 0.41% ) , pentadecane ( 0.58% ) , hexadecane ( 1.02% ) , heptadecane ( 0.53% ) , and cholesterol ( 5.07% ) ( table 2 ) . the analysis report reveals the presence of phthalate derivative and other minor volatile essential oils as potent antimicrobial agents extracted from the squid ink . marine natural products have been a strong source for novel drug products , or have been a model for introducing a commercial drug . squid ink is not the most elusive and enigmatic pigment found in nature but just a particle waiting for a rational study . in recent years , the problem of antimicrobial ( drug ) resistance is emerging and many diseases are increasingly difficult to treat because of the emerging drug - resistant organisms . the design of effective and novel dosing regimens that suppress the emergence and proliferation of resistant microbial populations is crucial . as resistance has increased to alarming proportion , a safe and cheaper source can always be an alternative to the routine therapeutics . as mollusk has been reported to possess various active molecules , the ink from the squid l. duvauceli previous associated studies state that the crude extraction of the squid ink is achieved using various solvents . successful prediction of natural bioactive molecules from natural sources is largely dependent on the type of solvent used in the extraction procedure and in many studies it was found that extracts in organic solvents provided more consistent antimicrobial activity . the crude extraction in our earlier studies is thus achieved by parallel solvent extraction method , where the ink is mixed with various solvents individually and is not added sequentially . tlc and hptlc analysis are the simplest and cheapest method of detecting any natural constituent because the method is easy to run and reproducible and requires little equipment . active crude extracts are chosen for column chromatography due to its relatively low complexity as seen with tlc , bioautography , and disc diffusion method . the solvent gradient used for elution of the biomolecules has been successfully standardized and is best achieved with n - hexane and ethyl acetate gradient . the selection of the solvent gradients can be rationalized in terms of the polarity of the compounds being extracted by each solvent and in addition to their intrinsic bioactivity , by their ability to dissolve or diffuse in the different media used in the assay . for the antimicrobial bioassay , the concentrated fractions were dissolved with dmso and were employed for the agar well diffusion bioassay . the choice of dmso as a solvent is due to its solvency for a wide range of chemicals , its low antibacterial activity at concentrations less than 2% , and its low toxicity . the findings of the antimicrobial bioassay report that the active fraction has antimicrobial efficacy against the gram positive cocci s. aureus , gram negative bacilli e. coli and k. pneumoniae , gram positive bacilli l. acidophilus , and the pathogenic yeast c. albicans . the active fraction scores a high activity against c. albicans that indicates its potent antifungal activity . the mbc value has also been deduced and is determined as 2.5 to 5 mg / ml against the tested organisms . microbial spot checker board also yields the same result with the absence of growth at the spot inoculated with the determined mbc value . gc - ms analysis of the bioactive fraction shows the presence of the bioactive compounds which are further confirmed with the library data . using mass spectroscopy the molecular mass of a compound and its elemental composition further this method involves very little amount of the test sample and gives the molecular weights accurately . gc - ms analysis has showed bis(2-ethylhexyl ) phthalate as the major constituent with large area normalization of 91.43% . it correlates with the mass spectrum of behp reported earlier from a phthalate isolated from a marine bacterial strain . the other minor constituents with low area normalization were also identified by the molecular mass from the library database . bis(2-ethylhexyl ) phthalate is found in low levels in the environment as it is subjected to biodegradation . this derivative has also been reported to be present in the fish and lipid tissues and also as a pollutant in marine environment . the bioactivity of the phthalate derivatives has already been reported in many plants , algae , and marine microorganisms and also from many marine species . few reports are available for the antibacterial potential of phthalate derivatives from plants and from flowers . bis(2-ethylhexyl ) phthalate extracted from streptomyces bangladheshiensis has been reported to be a potent antibacterial agent against gram positive bacteria . the other volatile minor compounds identified by gc - ms analysis have also been found to be potent antibacterial agents . the antibacterial activities of pentadecane and heptadecane compounds extracted from sea urchin have also been reported to possess potent activity against gram positive and gram negative bacteria . behp has been reported to possess a potent antifungal activity against major pathogenic fungi like candida , cryptococcus , and aspergillus sp . . the other minor constituents analyzed by gc - ms have also been reported to possess antifungal activity . naphthalene derivative has been reported to possess the same against c. albicans and aspergillus sp . . the antifungal activity of cholesterol hydrazone derivative has also been studied against c. albicans at a concentration of 1.5 g / ml . the antifungal activity of tetradecane and octadecane has been reported against c. albicans . gc - ms analysis of a natural cure concoction epa - ijebu showed the presence of natural alkanes such as hexadecane , heptadecane , and octadecane with potent antifungal activity . in correlation with these reports , the study results reveal that the ink has potent antimicrobial constituents which owes for its antibacterial and antifungal properties . the promising antimicrobial activity of these bioactive constituents needs a further multipronged research to implement its use as a novel therapeutic agent in near future for treating ailments with the drug resistant microbial pathogens . this study has suggested the presence of antimicrobial bioconstituents in the squid ink by column fractionation studies . gc - ms analysis has also aided the evaluation of the major and minor compounds present in it through its mass spectrum data . the study thus concludes the synergistic effects of an array of compounds in the squid ink towards its potent antimicrobial property . a novel therapeutic compound from a new marine source like the squid ink would be of much use in eradicating the microbial pathogens and it would definitely aid in the control and emergence of drug resistant strains . in recent years , drug - eluting stents ( des ) have been demonstrated to dramatically reduce the rate of restenosis and the need for repeat revascularization.1 - 3 ) despite these promising results , stent thrombosis seems to occur more frequently with des and often seems to be associated with clopidogrel resistance.4 ) we report a case of recurrent stent thrombosis associated with clopidogrel resistance in a patient with acute myocardial infarction . a 63-year - old female was transferred to the emergency department complaining of squeezing chest pain that had increased over the past twelve hours . she underwent emergent coronary angiography ( cag ) , which revealed critical stenosis in the proximal and middle left anterior descending coronary artery ( lad ) . she was successfully treated with two paclitaxeleluting stents ( 3.012 mm and 2.528 mm taxus stents , boston scientific , reading , pa , usa ) in the proximal and middle lad ( fig . she was treated with triple antiplatelet therapy ( aspirin 100 mg , clopidogrel 75 mg , and cilostazol 200 mg daily ) because she was found to be hyporesponsive to clopidogrel when tested for adenosine diphosphase ( adp)-induced platelet aggregation utilizing the verifynow p2y12 point - of - care assay ( 181/0 p2y12 reaction unit/% ) . three days after discharge , she again developed chest pain and presented to the emergency department with mental confusion associated with acute pulmonary edema . kinase - mb 34.0 u / l , troponin - i 67.01 ng / ml , troponin - t 5.5 ng / ml ) . emergency cag , after intubation due to hypoxemia , showed total occlusion of the mid - lad due to stent thrombosis ( fig . 4 ) . after intravenous administration of a glycoprotein iib / iiia receptor blocker ( reopro ) , balloon angioplasty was carried out multiple times using a 3.0 mm balloon at 10 - 12 atm because of recurrent , immediate thrombus formation and coronary occlusion . the next day , however , she complained of severe chest pain again , and the ecg showed marked st - segment elevation in v1-v6 , i , and avl ; and new - onset complete right bundle branch block with left anterior fascicular block ( fig . she was successfully treated with balloon angioplasty and a final angiogram revealed improved flow over stented lad without intraluminal filling defect . despite the cilostazol medication , adp - induced platelet aggregation showed that she was still hyporesponsive to clopidogrel ( 171/0 p2y12 reaction unit/% ) . she was discharged after uneventful recovery with triple anti - platelet therapy using an increased dose of aspirin ( aspirin 200 mg , clopidogrel 75 mg , cilostazol 200 mg daily ) . we report this case to draw more attention to stent thrombosis associated with clopidogrel resistance after des implantation . this case shows that a des patient with clopidogrel resistance can be vulnerable to stent thrombosis even if treated with triple anti - platelet therapy , which in recent studies has been shown to be more effective in preventing stent thrombosis than conventional dual anti - platelet therapy . stent thrombosis is an uncommon but serious complication of coronary artery stents that often presents as myocardial infarction ( mi ) or death . over several trials , the incidence of stent thrombosis was 0.58 - 1.3% in des.5)6 ) several factors have been associated with stent thrombosis , including older age , black race , diabetes mellitus , bifurcation lesion , in - stent restenosis lesion , procedure - related factors such as stent malposition , greater stent length , postprocedure acute renal failure , non - compliance to anti - platelet agent and anti - platelet resistance.7 - 9 ) anti - platelet resistance as an independent predictor of stent thrombosis , even several years after implantation of des , increases the risk of stent thrombosis . in this patient , stent thrombosis may have been caused by several risk factors , especially anti - platelet resistance . several therapeutic approaches ( the addition of cilostazol or a glycoprotein iib / iiia inhibitor , increased dosage of clopidogrel and aspirin ) might be taken for a patient with anti - platelet resistance . in our patient , although cilostazol ( 200 mg daily ) was added to conventional dual anti - platelet therapy , recurrent stent thrombosis occurred . subsequently , the daily dose of aspirin was increased from 100 mg to 200 mg . in the des era , stent thrombosis is a fatal complication and anti - platelet therapy has been shown to be very important in preventing stent thrombosis . thus , assessment of the patient 's responsiveness to anti - platelet agents may be a crucial factor in monitoring these drugs ' therapeutic efficacy and improving clinical outcomes after implantation of des . recent studies have shown that adequate anti - platelet effects are not achieved in 5% to 45% of the patients taking aspirin and in 4% to 30% of patients taking clopidogrel10)11 ) and therefore suggest that many patients are resistant or only partially responsive to the anti - platelet agents . currently , however , routine screening for anti - platelet resistance remains a persistent , unresolved issue and further evidence is necessary before it will be possible to recommend this testing as part of standard assessment of pci candidates . in addition , further prospective studies are needed to set guidelines for optimal treatment of patients with antiplatelet resistance who are at increased risk of stent thrombosis , a catastrophic complication of des implantation .

chromatographic characterization and the gc - ms evaluation of the black pigmented ink of loligo duvauceli in the present study have yielded an array of bioactive compounds with potent antimicrobial property . facing an alarm of antimicrobial resistance globally , a need for elucidating antimicrobial agents from natural sources will be the need for the hour . in this view , this study is aimed at characterizing the black pigmented ink of the indian squid l. duvauceli . the squid ink was subjected to crude solvent extraction and was fractionated by silica gel column chromatography . tlc and hptlc profiles were recorded . antimicrobial bioassay of the squid ink fractions was done by agar well diffusion method . the antimicrobial fraction was then characterized using gc - ms analysis . the results showed that the n - hexane extract upon column fractionation yielded a total of 8 fractions with the mobile phase of hex / etoac in different gradients . tlc and hptlc profiles showed a single spot with a retention factor of 0.76 . fraction 1 showed significant antibacterial activity against escherichia coli , klebsiella pneumoniae , staphylococcus aureus , and lactobacillus acidophilus and a promising antifungal activity against candida albicans . the antimicrobial fraction upon gc - ms analysis of bis(2-ethylhexyl ) phthalate ( behp ) possesses the highest percentage of area normalisation ( 91% ) with other few minor constituents . the study is concluded by stating that the antimicrobial efficacy of the squid ink might be due to the synergistic effects of the phthalate derivative and the other minor volatile compounds analysed in the squid ink . drug - eluting stents ( des ) are considered the treatment of choice for most patients with obstructive coronary artery disease when percutaneous intervention ( pci ) is feasible . however , stent thrombosis seems to occur more frequently with des and occasionally is associated with resistance to anti - platelet drugs . we have experienced a case of recurrent stent thrombosis in a patient with clopidogrel resistance . a 63-year - old female patient suffered from acute myocardial infarction and underwent successful pci of the left anterior descending coronary artery ( lad ) with two dess . she was found to be hyporesponsive to clopidogrel and was treated with triple anti - platelet therapy ( aspirin 100 mg , clopidogrel 75 mg , and cilostazol 200 mg daily ) . three days after discharge , she developed chest pain and was again taken to the cardiac catheterization laboratory , where coronary angiography ( cag ) showed total occlusion of the mid - lad where the stent had been placed . after intravenous administration of a glycoprotein iib / iiia inhibitor , balloon angioplasty was performed , resulting in thrombolysis in myocardial infarction ( timi ) iii antegrade flow . the next day , however , she complained of severe chest pain , and the electrocardiogram showed marked st - segment elevation in v1-v6 , i , and avl with complete right bundle branch block . emergent cag revealed total occlusion of the proximal lad due to stent thrombosis . she was successfully treated with balloon angioplasty and was discharged with triple anti - platelet therapy .

abnormal knee kinematics are considered to be an important factor influencing the long - term outcomes after cruciate ligament injuries1,2,3 . in order to improve long - term outcomes of treatments for the patients with cruciate ligament injuries , therefore , it is important to precisely evaluate the kinematics of knees with cruciate ligament injuries . the motion of a knee joint with ligament injury is commonly evaluated by static methods such as the lachman test , posterior drawer test , valgus stress test , and arthrometric measurements , e.g. kt-1000 . however , patients with knee ligament deficiencies usually complain about their symptoms under dynamic conditions . therefore , there is a possibility that the instability of the knee joint evaluated in static conditions does not reflect the functional disabilities of its ligament injury1 , 3,4,5 . three - dimensional motion analysis using optical skin marker systems has been widely used as an evaluation method of body kinematics in dynamic conditions with muscular activations . this approach provides six - degrees - of - freedom motion of the kinematics of the knee during activities of daily living without restricted conditions in vivo . andriacchi et al . developed a point cluster technique ( pct ) , which employs an overabundance of markers ( a cluster ) placed on each segment to minimize the effects of skin movement artifacts6 . the pct can be extended to minimize skin movement artifact by optimal weighting of the markers according to their degree of deformation . the criteria of the normal range of three - dimensional knee kinematics during walking remain uncertain . side - to - side difference in the range of knee motion is widely used as a criterion of the normal range of knee motion1 . to detect an abnormal knee motion during walking , it is useful to determine the normal range of the side - to - side difference in knee kinematics during walking based on the data using the pct . to the best of our knowledge , however , no study has examined this ; therefore , the purpose of the present study was to determine the normal range of the side - to - side difference in knee kinematics using pct during walking based on three dimensional kinematics of the knee of healthy subjects . the study was approved by the research ethics committee of hakodate national college of technology and informed consent was obtained from each subject before the test . twenty - one healthy normal volunteers without knee pain or an episode of injury to the legs were recruited for the study . the subjects were 11 males and 10 females with an average age of 27.0 ( sd 7.2 ) years , and ranging from 21 to 47 years , and an average body mass index of 20.6 ( sd 2.4 ) kg / m , ranging from 16.8 to 27.7 kg / m . the subjects were tested bilaterally at a self - selected normal walking speed during their walking for 8 m after the first four strides . the subjects walked barefoot on a hard surface at an average walking speed of 1.25 m / s . the subjects took a rest before each trial until they had recovered from fatigue . the kinematic data were collected by a three - dimensional motion analysis system using four infrared light cameras ( rrorflex , qualisys ab inc . , ground reaction forces during the tests were also measured using two multi - component force plates ( or6 , advanced mechanical technology inc . , three - dimensional motion data were processed using qualisys track manager ( qualisys track manager 3d , qualisys ab inc . , force data were used to identify the time of heel strike in each gait cycle . a gait cycle was defined as the interval between heel strike to heel strike on the ipsilateral side . light - reflective markers were placed by physical therapists well - trained in the pct and body surface anatomy6 . kinematic measurements were made of 21 light - reflective markers arranged on two limb segments , creating separate cluster groups of 11 markers on the thigh and 10 markers on the shank . the pct employs an overabundance of markers ( a cluster ) placed on each segment to minimize the skin motion artifact due to segmental form change by muscle contraction and marker - oscillation6 . the pct is based on a cluster of points uniformly distributed on the limb segment . each point is assigned an arbitrary mass , which can be varied at each time step . the center of mass and the inertia tensor of the cluster of points are calculated as described below . the eigenvalues and the eigenvectors of the inertia tensor are the principal moments of inertia and the principle axes of the point cluster . the eigenvalues are invariant to movement if the segment is behaving as a rigid body . if the eigenvalues change from their value in the rest position , then the segment where the markers are placed has deviated from rigid body movement . the individual weighting is adjusted by redistributing the weight factors at each time step to minimize the eigenvalue changes . examined the pct using simulation trials with systematic and random components of deformation error introduced into marker position vectors7 . they also tested the pct in vivo with a subject fitted with an external fixation device ( ilizarov ) . the average location and orientation measurement errors of the pct were 0.6 mm ( sd 0.6 mm , maximum approximately 3 mm ) and 0.5 degrees ( sd 0.5 degrees , maximum approximately 4 degrees ) respectively7 . another study has also reported an accuracy of 0.79 degrees ( sd 0.48 degrees ) for flexion - extension , 1.79 degrees ( sd 0.93 degrees ) for internal - external rotation , and 0.40 degrees ( sd 0.12 degrees ) for abduction - adduction8 . intra - class correlations coefficients ( 1 , 5 ) and standard errors of measurement of the pct have been calculated using the data of a previous study and indicate substantial or almost perfect reliability9 . in the present study , the position coordinate data were converted to flexion - extension ( fe ) , abduction - adduction ( aa ) , internal - external ( ie ) rotation , and the anterior - posterior ( ap ) , medial - lateral ( ml ) , superior - inferior ( si ) translation of the tibia with respect to a gait cycle6 . data from three trials were averaged and the average was used for the analysis of each limb . the value of the right leg subtracted from that of the left leg was defined as the side - to - side difference in rotation / translation in each stage of the gait cycle . we then determined the average side - to - side differences and their 95% confidence intervals ( cis ) . for statistical analysis , one - way analysis of variance and post - hoc bonferroni tests were performed to compare the average side - to - side difference of each motion among 0% , 20% , 40% , 60% and 80% of the time of a gait cycle . differences were considered statistically significant at p < 0.05 with bonferroni adjustment for post - hoc multiple comparisons . based on the ground reaction forces of the force plates , the stance and swing phases during walking were determined . as a result , 0% to mean 61.6% ( sd 1.3% ) of the gait cycle was regarded as the stance phase . therefore , 0% , 20% , 40% , 60% and 80% of the gait cycle were considered as the early stance phase , the middle stance phase , the late stance phase , the early swing phase and the late middle swing phase , respectively . the average knee kinematics pattern of the right and left legs during a gait cycle are shown in fig . 1.the average knee kinematics pattern of the right and left legs during a gait cyclea ) flexion - extension ; b ) adduction - abduction ; c ) internal - external rotation of the tibia . regarding fe of the knee during walking , the average values ( 95% cis ) of side - to - side differences at each time frame of normal walking were 4.3 degrees ( 3.0 to 5.7 degrees ) , 4.9 degrees ( 3.5 to 6.3 degrees ) , 4.4 degrees ( 2.9 to 5.9 degrees ) , 7.4 degrees ( 5.2 to 9.6 degrees ) and 5.4 degrees ( 3.4 to 7.3 degrees ) at 0% , 20% , 40% , 60% , and 80% of the gait cycle , respectively ( fig . 2.the 95% confidence intervals ( cis ) of side - to - side differences of the normal subjectsa ) flexion - extension ( fe ) ; b ) abduction - adduction ( aa ) ; c ) internal - external ( ie ) rotation ; d ) anterior - posterior ( ap ) translation ; e ) medial - lateral ( ml ) translation ; f ) superior - inferior ( si ) translation , table 1table 1.the average of side - to - side differences and their 95% confidence intervals in each stage of the gait cyclewalking cycle0%20%40%60%80%rotation ( degrees)flexion - extension4.3 ( 3.05.7)4.9 ( 3.56.3)4.4 ( 2.95.9)7.4 ( 5.29.6)5.4 ( 3.47.3)abduction - adduction3.8 ( 2.65.1)4.5 ( 2.76.4)3.2 ( 2.14.3)5.4 ( 3.67.2)8.9 ( 5.911.9)internal - external7.4 ( 5.59.4)5.7 ( 3.77.8)4.7 ( 3.36.1)6.2 ( 4.08.4)7.4 ( 4.710.2)translation ( mm)anterior - posterior9.7 ( 6.712.7)7.7 ( 4.510.9)6.8 ( 4.19.6)9.2 ( 5.013.4)12.8 ( 6.818.8)medial - lateral6.3 ( 3.79.0)5.3 ( 3.47.1)5.5 ( 3.77.3)6.7 ( 4.98.5)5.9 ( 3.78.1)superior - inferior7.7 ( 4.510.8)8.1 ( 4.211.9)8.2 ( 4.112.4)8.9 ( 4.413.5)9.8 ( 9.818.8)(mean , 95% ci ) . a : post - hoc bonferroni tests ( vs. 0% : p<0.001 , vs. 20% : p<0.001 , vs. 40% : p<0.001 , vs. 60% : p<0.001 ) . b : post - hoc bonferroni test ( vs. 40% p<0.001 ) ) . there were no significant differences in the side - to - side differences of fe among 0% , 20% , 40% , 60% , and 80% of the gait cycle ( p=0.086 ) . the average side - to - side differences ( 95%cis ) of aa were 3.8 degrees ( 2.6 to 5.1 degrees ) , 4.5 degrees ( 2.7 to 6.4 degrees ) , 3.2 degrees ( 2.1 to 4.3 degrees ) , 5.4 degrees ( 3.6 to 7.2 degrees ) and 8.9 degrees ( 5.9 to 11.9 degrees ) at 0% , 20% , 40% , 60% , and 80% of the gait cycle , respectively ( fig . the side - to - side difference of aa at 80% of the walking cycle was significantly greater than those at 0% , 20% , 40% and 60% of the gait cycle ( vs. 0% : p<0.001 , vs. 20% : p<0.001 , vs. 40% : p<0.001 , vs. 60% : p<0.001 ) . the average side - to - side differences ( 95%cis ) of ie rotation were 7.4 degrees ( 5.5 to 9.4 degrees ) , 5.7 degrees ( 3.7 to 7.8 degrees ) , 4.7 degrees ( 3.3 to 6.1 degrees ) , 6.2 degrees ( 4.0 to 8.4 degrees ) and 7.4 degrees ( 4.7 to 10.2 degrees ) at 0% , 20% , 40% , 60% , and 80% of the gait cycle , respectively ( fig . there were no significant differences in the side - to - side differences of ie rotation among 0% , 20% , 40% , 60% , and 80% of the gait cycle ( p=0.332 ) . at 0% , 20% , 40% , 60% , and 80% of the gait cycle , the average values ( 95% cis ) of side - to - side differences of ap translation were 9.7 mm ( 6.7 to 12.7 mm ) , 7.7 mm ( 4.5 to 10.9 mm ) , 6.8 mm ( 4.1 to 9.6 mm ) , 9.2 mm ( 5.0 to 13.4 mm ) and 12.8 mm ( 6.8 to 18.8 mm ) , respectively ( fig . the side - to - side difference of ap translation at 80% of the gait cycle was significantly greater than that at 40% of the gait cycle ( p=0.001 ) . the average side - to - side differences ( 95%cis ) of ml translation were 6.3 mm ( 3.7 to 9.0 mm ) , 5.3 mm ( 3.4 to 7.1 mm ) , 5.5 mm ( 3.7 to 7.3 mm ) , 6.7 mm ( 4.9 to 8.5 mm ) and 5.9 mm ( 3.7 to 8.1 mm ) , respectively ( fig . 2-e , table 1 ) . there were no significant differences in the side - to - side differences of ml translation among 0% , 20% , 40% , 60% , and 80% of the walking cycle ( p=0.875 ) . similarly the average side - to - side differences ( 95%cis ) of si translation were 7.7 mm ( 4.5 to 10.8 mm ) , 8.1 mm ( 4.2 to 11.9 mm ) , 8.2 mm ( 4.1 to 12.4 mm ) , 8.9 mm ( 4.4 to 13.5 mm ) and 9.8 mm ( 9.8 to 18.8 mm ) , respectively ( fig . there were no significant differences in the side - to - side differences of sl translation among 0% , 20% , 40% , 60% , and 80% of the walking cycle ( p=0.947 ) . the average knee kinematics pattern of the right and left legs during a gait cycle a ) flexion - extension ; b ) adduction - abduction ; c ) internal - external rotation of the tibia the 95% confidence intervals ( cis ) of side - to - side differences of the normal subjects a ) flexion - extension ( fe ) ; b ) abduction - adduction ( aa ) ; c ) internal - external ( ie ) rotation ; d ) anterior - posterior ( ap ) translation ; e ) medial - lateral ( ml ) translation ; f ) superior - inferior ( si ) translation ( mean , 95% ci ) . a : post - hoc bonferroni tests ( vs. 0% : p<0.001 , vs. 20% : p<0.001 , vs. 40% : p<0.001 , vs. 60% : p<0.001 ) . b : post - hoc bonferroni test ( vs. 40% p<0.001 ) for clinical application , the knee kinematics of a case with the posterior cruciate ligament injury ( pcl ) were also evaluated . a 37 year - old male suffered a knee injury while playing rugby football . the case showed grade-3 positive in the posterior drawer test , and posteromedial rotatory instability11 . the mri images indicated a pcl injury . at two months after injury , evaluation of 3-d kinematics during walking based on the side - to - side differences of kinematic data during each stage of gait , the tibia of the injured leg was shifted posteriorly in ap translation during 20% to 60% walking cycle , and shifted internally in ie rotation during 0% to 60% gait cycle ( fig . 3fig . 3the side - to - side difference of the femur relative to the tibia in the case with pcl and pms injuries with the 95% confidence intervals ( cis ) of the normal subjects . a ) anterior - posterior ( ap ) translation ; b ) internal - external ( ie ) rotation ) . the side - to - side difference of the femur relative to the tibia in the case with pcl and pms injuries with the 95% confidence intervals ( cis ) of the normal subjects . a ) anterior - posterior ( ap ) translation ; b ) internal - external ( ie ) rotation the present study investigated the side - to - side differences in kinematics of the knee joint during walking by healthy subjects . the side - to - side differences in adduction - abduction rotation and anterior - posterior translation of the tibia were found to be highly dependent on the stage of gait , and they were significantly larger in the swing phase than in the stance phase . therefore , our findings suggest that it is important to know the normal ranges of the side - to - side differences in knee kinematics in each stage of the gait cycle , in particular those of adduction - abduction and anterior - posterior translation of the knee , in order to evaluate the knee kinematics of subjects during walking . the reason why the side - to - side differences of adduction - abduction rotation and anterior - posterior translation of the tibia during walking are larger in the swing phase than in the stance phase remain unknown . one possible reason is mechanical restraint of the motion of the tibia during walking . the leg is not subject to weight bearing in the swing phase , and the tibia can move with less mechanical restraint in the swing phase than in the stance phase . without weight bearing , the knee is relaxed and the joint kinematics are likely to be influenced by ligament balance , rather than joint morphology . the knee kinematics during walking of a pcl injury case are also presented . for this case , an abnormal side - to - side difference in internal rotation of the tibia during walking as well as an abnormal side - to - side difference in posterior translation of the tibia were found . li et al . reported that pcl deficiency significantly increases posterior translation of the tibia during knee flexion under a weight - bearing condition using a dual - orthogonal fluoroscopic system12 . however , there was no significant difference in internal - external rotation between pcl - injury and normal knees throughout the range of flexion . in our present study , the case might have had associated injuries of posteromedial structures of the knee . previous cadaver studies have shown that additional injuries of posteromedial structures significantly increase internal rotation of the tibia as well as posterior translation of the tibia in pcl deficient knees13,14,15 . regarding clinical relevance , our findings indicate that the evaluation of the side - to - side difference of knee - joint kinematics provides useful information for clinical practice . however , caution is required in the clinical application of the evaluation of the side - to - side difference of knee - joint kinematics during walking because , there is a possibility that the contralateral leg would functionally adapt to the condition after trauma or disease . some investigators have reported that the contralateral leg as well as the injured leg functionally adapt to the condition after the injury of the acl and show the so - called quadriceps - avoidance gait16,17,18,19 . therefore , the evaluation of the side - to - side difference of knee joint kinematics may underestimate the knee kinematics of an injured leg . while this approach can measure in vivo kinematics of the knee in a dynamic condition , namely in a more physiological condition , it has been reported to have low accuracy due to skin movement artifacts20 , 21 . the recently described pct employs an overabundance of markers ( a cluster ) placed on each segment to minimize the effects of skin movement artifact6 . the pct can be extended to minimize skin movement artifacts by optimal weighting of the markers according to their degree of deformation6 . furthermore pct permits direct in vivo measurement of the complete six - degrees - of - freedom motion of the kinematics of the knee during activities of daily living . pct has , therefore , recently attracted attention , since the data of three dimensional kinematics of the knee during activities of daily living including walking are considered to reflect function of the knee joint . walking speed , surface condition , and shoes presumably influence the results of gait analysis . the knee kinematics measured in this study during walking and gait speed were similar to those reported by other investigators22,23,24,25,26 . first , since the point cluster technique is based on skin makers , the accuracy of the relative positions of the femur with respect to the tibia depends on the marker attachment technique of the examiner . second , the sample size of the present study , 21 , was not large enough to determine the normal range of the side - to - side - difference of the knee kinematics during walking . third , the effect of age or gender differences was not investigated in the present study . however , no significant differences have been reported between age groups and gender in previous knee kinematics studies27,28,29 , suggesting their effects in this study would have been small . despite the above - noted limitations , the mean side - to - side differences and their 95% cis as objective criteria of normal knee kinematics are useful information for the functional evaluation of subjects with disorders of the knee joint . in conclusion , the knee kinematics of healthy subjects during walking were measured using the pct , and the normal range of side - to - side differences in knee kinematics were determined for five stages of the gait cycle . the side - to - side differences in aa rotation and ap translation were highly dependent on the stage of gait ; therefore , the normal ranges of the side - to - side differences in knee kinematics of each stage of gait , in particular aa rotation and ap translation of the tibia , are useful information for the evaluation of the normal and pathological knee kinematics during walking . alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) receptor channels mediate most of the fast synaptic excitatory transmission in the brain . importantly , the glua2 subunit with its edited q / r site ( sommer et al . , 1992 ) ensures that ampa receptors containing this subunit are largely ca - impermeable ( hollmann et al . , 1991 ; burnashev et al . , 1992 ; jonas and burnashev , 1995 ; isaac et al . , 2007 ) . by contrast , glua2-lacking ampa receptor channels , i.e. , any homomeric or heteromeric assembly of glua1 , a3 , or a4 , are ca - permeable and subject to voltage - dependent block by intracellular polyamines , resulting in a characteristic doubly rectifying current - voltage ( i v ) relationship . imbalance in favor of ca permeable ampars can cause cns dysfunction ( liu and zukin , 2006 ) . in synaptic ampa channels , sensitivity to endogenous polyamines and the degree of rectification can be lessened by interaction with tarps , the transmembrane ampar regulatory proteins ( soto et al . , 2007 ) . thus , a low extent of rectification of synaptic ampar - mediated currents need not reflect a particular ampar composition , but may also indicate interaction with regulatory factor(s ) . as gleaned from the properties of ampa receptors in different neuronal populations , the current consensus would state that principal excitatory neurons operate with glua2-containing ampa receptors ( adesnik and nicoll , 2007 ; lu et al . , 2009 ) , whereas gaba - ergic inhibitory neurons often additionally feature glua2-lacking synaptic ampa receptors ( geiger et al . , 1995 ; toth and mcbain , 1998 ; liu and cull - candy , 2002 ) . while there is firm consensus , 1992 ; lu et al . , 2009 ) , conflicting evidence surrounds the possible presence of glua2-lacking ampar channels in hippocampal schaffer collateral / commissural synapses in ca1 pyramidal cells , arguably the best - studied synapses in the brain . several groups observed that the molecular identity of ampars in ca1 cell synapses is altered upon induced changes in synaptic strength . plant et al . ( 2006 ) reported that induction of long - term potentiation ( ltp ) elicits a transient increase in the proportion of polyamine sensitive ampars at the potentiated synapses , revealed by an increased rectification index ( ri ) . , adesnik and nicoll ( 2007 ) failed to obtain evidence for the insertion of synaptic ca - permeable ampa receptors upon ltp induction , concluding that all synaptic ampars in ca1 cells contain glua2 subunits . thus , whether glua2-lacking ampars exist at glutamatergic ca1 cell synapses and contribute to fast transmission clearly needs re - evaluating . the most common approach to detect the participation of glua2-lacking ampars in synaptic transmission is to test sensitivity to the wasp polyamine toxin , philanthotoxin-433 ( phtx-433 ) . glua2-lacking channels can be strongly blocked by phtx-433 in an activity dependent manner , while glua2 containing ampars are insensitive to the drug ( washburn and dingledine , 1996 ) . however , bath - applied phtx-433 may target a glua2-lacking channel population already blocked by endogenous polyamines ( pa ) , and thus , a moderate contribution of glua2-lacking channels could be overlooked . in this context , it is important to note that virtually all glua2-lacking ampar channels contain polyamine in the closed state , regardless of the membrane potential . this results in the characteristic doubly rectifying shape of their i v curves , which reflects the speed of relief from polyamine block . this speed is dependent on the absolute transmembrane electric field , and is faster when the cell is hyperpolarized ( rozov et al . , 1998 ) . even at physiological resting membrane potentials ( 70 mv ) , a substantial portion of glua2-lacking channels could still be blocked by polyamines during single unitary epscs , and repetitive high - frequency stimulation is required to relieve the block ( rozov and burnashev , 1999 ) . hence , to get an accurate readout of the phtx effect on ampar - mediated currents the influence of endogenous polyamines should first be minimized . to resolve the issue of ca - permeable ampa channels in ca1 pyramidal cells of wild - type ( wt ) , we took recourse to gene - targeted mice lacking either glua2 ( shimshek et al . , 2006 ) or glua1 ( zamanillo et al . , 1999 ) or expressing glua1 in a q / r site - edited version ( sanchis - segura et al . , 2006 ) . this allowed us to analyze the artificial situations of ca1 cell synapses having ampa receptors of maximal ( glua2 mutant mice ) or minimal ( glua1 and glua1(r ) mutants ) sensitivity to polyamines . the presence of ca - permeable ampa channels in wt should then be revealed by increased sensitivity to phtx-433 relative to ampa channels in glua1 and glua1(r ) mouse mutants . no such increase in phtx-433 sensitivity should be observed if wt ca1 pyramidal cell synapses indeed lack ca - permeable ampa channels , as posited by a prevalent view ( adesnik and nicoll , 2007 ) . our data clearly demonstrate that in wt ca1 synapses , 810% of the ampa channels are ca - permeable . transverse hippocampal 250 m slices were prepared from the brains of 4256 day - old wt , glua1 ( zamanillo et al . , 1999 ) , glua1(r ) ( sanchis - segura et al . , 2006 ) and glua2 mice ( shimshek et al . , 2006 ) , killed by decapitation . the slicing chamber contained an oxygenated ice - cold solution [ modified from ( dugue et al . , 2005 ) ] composed of ( in mm ) : k - gluconate , 140 ; n-(2-hydroxyethyl ) piperazine - n-ethanesulfonic acid ( hepes ) , 10 ; na - gluconate , 15 ; ethylene glycol - bis ( 2-aminoethyl)-n , n , n,n-tetraacetic acid ( egta ) , 0.2 ; and nacl , 4 ( ph 7.2 ) . slices were incubated for 30 min at 35c before being stored at room temperature in artificial csf ( acsf ) containing ( in mm ) : nacl , 125 ; nahco3 , 25 ; kcl , 2.5 ; nah2po4 , 1.25 ; mgcl2 , 1 ; cacl2 , 2 ; and glucose , 25 ; bubbled with 95%o2 and 5%co2 . patch electrodes were pulled from hard borosilicate capillary glass ( sutter instruments flaming / brown micropipette puller ) . these were filled with either a polyamine - free or a polyamine - containing solution . the polyamine - free solution consisted of ( in mm ) cs - gluconate , 100 ; cscl , 10 ; hepes , 10 ; nacl , 8 ; egta , 0.2 ; mgatp , 2 ; na3gtp , 0.3 ; phosphocreatine , 10 ; and k2atp , 20 ( ph 7.3 with csoh ) . the polyamine - containing solution was identical except for the addition of 0.5 mm spermine , yielding a free spermine concentration of 40 m ( watanabe et al . , 1991 ) . whole - cell recordings from these neurons were made in voltage - clamp mode using a heka epc-9 amplifier ( list elektronik ) . glass electrodes filled with acsf were placed in the dendritic region of stratum radiatum , 50100 m from the cell body , to stimulate schaffer collateral inputs at inter - stimulus intervals of 6 s. the stimulation intensity was adjusted to produce an epsc with an amplitude of 50 pa at the beginning of each recording . inhibitory synaptic transmission was blocked during recordings by adding 10 m gabazine to the perfusion acsf . for analysis , five subsequent responses were averaged and normalized to the mean epsc amplitude obtained during 2030 min of recording . the degree of blockade was calculated as a ratio of the average steady - state current amplitude before and after phtx-433 application . series resistance was monitored , and data from cells in which series resistance varied by > 15% during recording were discarded from analysis . glutamate ( 1 mm ) was applied using a piezo - controlled ( p 245.70 , physik instrumente , waldbronn , germany ) fast application system with a double - barrel application pipette onto outside - out patches of hek293 cells constitutively expressing homomeric glua1 receptors . for statistical analysis , student 's t - test was used , and data are presented as mean sd . we compared the effect of phtx-433 on epscs evoked in ca1 pyramidal cell synapses in hippocampal slices of glua2 mice in two distinct experimental conditions , one ensuring chelation of intracellular polyamines and the other having buffered spermine added to the intracellular solution . as in this mouse mutant all ampa channels are polyamine sensitive , we could evaluate the maximal effect of the applied concentration of phtx-433 ( 10 m ) on epscs mediated solely by glua2-lacking synaptic ampa channels . this should be useful for revealing their existence and estimating their contribution in wt , in which they would co - exist with glua2-containing ampars . additionally , we evaluated the sensitivity of synaptic ampars to intracellular polyamines in glua2 mice and compared it to that of wt . to this end , we measured the synaptic current - voltage ( i v ) relationship in ca1 pyramidal neurons dialyzed with polyamine - containing intracellular solution . evoked epscs from patched ca1 pyramidal cells of glua2 mice were recorded by stimulating schaffer collaterals at interstimulus intervals of 6 s with either polyamine - containing or -free patch - pipette solution . the washout of endogenous polyamines led to a three to fourfold increase in epsc amplitudes ( 0.29 0.27 relative to the steady - state values ; figures 1a , b ) . subsequent application of phtx-433 ( 10 m ) reduced the amplitude of responses to 0.34 0.15 compared to maximal amplitudes after polyamine washout ( n = 7 from 4 mice ; p < 0.001 ) . thus , 10 m of phtx-433 blocked 70% of the glua2-less ampa channels . however , when 0.5 mm of spermine was included in the pipette solution we typically observed a slight , though not significant , reduction in the epsc amplitude as whole - cell dialysis was progressing . initial amplitudes were 1.12 0.28 relative to the steady - state level reached after 30 min of dialysis ( p > 0.05 ; n = 7 from 5 mice ) . reduction of postsynaptic responses by subsequently applied phtx-433 was significantly less pronounced when compared to polyamine - free conditions ( 0.63 0.09 ; p < 0.01 ; figures 1a , b ) . maximal and polyamine - attenuated levels of phtx-433 blockade in ca1 pyramidal cells of glua2 mice . ( a ) the time course of epsc amplitude changes during washout of polyamine and subsequent phtx-433 ( 10 m ) application , when ca1 pyramidal neurons were dialyzed with polyamine - free intracellular solution ( green circles ) . also shown is the effect of exogenously added spermine on epsc amplitudes and the masking effect of spermine on phtx-433 suppression ( black circles ) . ( b ) representative averaged traces at the indicated times of recording in polyamine - free conditions are shown in green , in polyamine - containing condition in black . ( c ) effect of intracellular polyamines on ampar - mediated synaptic currents in glua2 and wt mice . ampar - mediated synaptic i v curves measured with polyamine - containing intracellular solution in glua2 and wt mice ( left panel ) . rectification properties of synaptic ampar channels in glua2 and wt mice ( right panel ) . ris were calculated as the ratio of epsc amplitudes measured at 30 and 70 mv ( epsc70/epsc30 ) . collectively , the data from glua2 mice suggest that endogenous or exogenously added polyamines constitutively block glua2-lacking channels and attenuate the effect of bath - applied phtx-433 . strong sensitivity of ampars to polyamines in glua2 mice was further demonstrated by measuring the synaptic i blockade in these mutants of the glua2-less ampars by polyamines resulted in a characteristic inwardly rectifying i figure 1c compares the rectification properties of ampar channels mediating synaptic transmission at schaffer collateral / commissural synapses in ca1 pyramidal cells of glua2 and wt mice . obviously , glua2 ablation leads to enhanced polyamine sensitivity relative to wt , as reflected by an increased ri ( glua2 : ri = 10.3 0.85 , n = 5 ; wt : ri = 3.9 0.77 n = 5 ; p < 0.01 ) . we next determined how the duration of agonist application affects the phtx-433 blocking potency and to what extent the current suppression by the toxin depends on intracellular polyamine content . we employed fast agonist application on outside - out patches of hek293 cells expressing homomeric glua1 receptor channels in presence of 50 m cyclothiazide to exclude an influence of channel desensitization . application of 1 mm glutamate for 100 ms at 70 mv evoked stable inward currents , which were drastically reduced in presence of 10 m phtx-433 , reaching a steady - state level of 3 1% of the initial amplitude ( figure a1a ; however , at 5 ms agonist applications , the steady - state level reached upon phtx-433 application was significantly higher ( 28 11% of control amplitudes ; figure a1b ; n = 5 ; p < 0.01 ) . similar experiments with a polyamine - free pipette solution demonstrated a reduction of glutamate - evoked currents ( 5 ms ; 1 mm ) that was stronger ( 11 7% relative to control values ; figure a1c ; n = 5 ) than the 28 11% observed with the pa - containing solution ( p < 0.05 ) . thus , usage of phtx-433 as a tool to probe the expression of glua2-lacking ampars requires the washout of endogenous polyamines . we performed similar experiments on glua1 and glua1(r ) mouse mutants , in which the likelihood for the presence in ca1 pyramidal cells of synaptic glua2-less , ca - permeable ampa channels is low and hence , sensitivity to polyamines and phtx-433 should be lacking . indeed , prolonged whole - cell dialysis with polyamine - free intracellular solution did not result in a significant increase in epsc amplitudes in ca1 pyramidal cells in either glua1 or glua1(r ) mice . we did record a moderate enhancement of ampar - mediated responses ( 0.78 0.1 and 0.81 0.11 , n = 7 cells , for 45 mice of each mutant line ) , which is most likely due to frequency facilitation or augmentation ( thomson , 2000 ; zucker and regehr , 2002 ) . as expected , application of phtx-433 ( 10 m ) did not affect epsc amplitudes in the glua1 ( 0.98 0.13 ; n = 7 from 4 mice ) or glua1(r ) mutants ( 1.07 0.16 ; n = 7 from 5 mice ) . synaptic ampar channels in ca1 pyramidal cells of glua1 and glua1(r ) mouse mutants are polyamine and phtx-433 insensitive . ( a ) the time course of epsc amplitude changes recorded in ca1 pyramidal neurons of glua1 ( green circles ) and glua1(r ) ( blue circles ) mutant mice during washout of polyamine and subsequent phtx-433 ( 10 m ) application . ( b ) representative averaged traces at the indicated times of recording from glua1 mice shown in green , and for glua1(r ) mutants in blue . ( c ) effect of intracellular polyamines on rectification properties of ampar - mediated synaptic currents in glua1 and wt animals . synaptic ampar - mediated i v curves measured with polyamine - containing intracellular solution in glua1 and wt mice ( left panel ) . rectification indices of synaptic ampar channels in glua2 and wt mice ( right panel ) . addition of 0.5 mm spermine to the intracellular solution did not cause any inward rectification of ampar - mediated synaptic currents in glua1 mice , and the i v curve became slightly outwardly rectifying ( figure 2c ) . however the shape of the i v curve and the ri measured in glua1 mice differed significantly from those in wt mice under the same experimental conditions ( figure 2c ; glua1 : ri = 2.32 0.42 , n = 5 and wt : ri = 3.9 0.77 n = 5 ; p < 0.05 ) . we conclude that absence of glua1 ( or expression of glua1 in its r - form ) greatly diminish the presence of synaptic ca - permeable ampa channels . according to previous studies , epscs recorded from ca1 pyramidal cells are insensitive to phtx-433 ( adesnik and nicoll , 2007 ; lu et al . , 2009 ) . as intracellular polyamines can occlude block by phtx-433 ( figure 1 ) , we first determined how endogenous polyamine washout changes the potency of exogenously applied phtx-433 on epsc amplitudes in wt ca1 cells . when the cells were dialyzed with polyamine - free solution , the amplitudes of evoked ampar - mediated responses increased during the first 1520 min of recording , finally reaching a steady - state level ( figures 3a , b ) . n = 9 from 6 mice ) , when normalized to the steady - state values ( 2030 min of recording ) . subsequent application of phtx-433 ( 10 m ) caused a gradual reduction of the postsynaptic responses , reaching a plateau level ( 5060 min of recording ) of 0.69 0.17 ( p < 0.01 ) , as compared to values prior to phtx-433 application ( figures 3a , b ) . ( a ) scatter plot shows epsc amplitude dynamics during prolonged whole - cell dialysis and following phtx-433 ( 10 m ) application , when ca1 pyramidal neurons were patched with either polyamine - free ( green circles ) or polyamine - containing ( black circles ) intracellular solution . ( b ) representative averaged traces at the indicated times of recording in polyamine - free ( green ) or polyamine - containing ( black ) solution . ( c ) dependence of ampar synaptic channel rectification properties in wt ca1 pyramidal cells on intracellular polyamine content . synaptic ampar - mediated i v curves measured with polyamine - containing and -free intracellular solutions ( left panel ) . dependence of rectification indices of synaptic ampar channels on the presence of polyamines in wt mice ( right panel ) . by contrast , when wt ca1 neurons were patched with polyamine - containing solution , the amplitudes of responses during the initial phase of dialysis were either stable or underwent a slight , but not significant reduction ( 1.18 0.14 , p > 0.05 , n = 7 from 5 mice ) . moreover , application of phtx-433 caused no significant changes in epsc amplitudes ( 1.12 0.14 ; p > 0.05 ) . thus , in wt ca1 pyramidal cells , washout of intracellular polyamines resulted in an increase in the amplitude of evoked postsynaptic responses , which was in large part counteracted by subsequent application of phtx-433 ( figures 3a , b ) . this remarkable difference to the results from synaptic ampars in ca1 pyramidal cells of glua1 and glua1(r ) mouse mutants constitutes unequivocal evidence for the presence of synaptic ca - permeable ampa receptors in wt ca1 cells . to further substantiate our finding on the existence of glua2-lacking ampars at schaffer collateral / commissural synapses in wt ca1 pyramidal cells , we evaluated the effect of the presence of intracellular spermine on the shape of ampar - mediated i v curves and on the ri . v relation recorded with polyamine - free solution was nearly linear and deviated strongly at positive potentials from that observed with polyamine - containing solution . also , the ri in presence of spermine was significantly higher compared to that in polyamine - free conditions [ wt ( sp ) : 3.9 0.77 , n = 5 and wt ( no sp ) : 2.86 0.28 , n = 5 ; p < 0.05 ] . we compared the effect of phtx-433 on epscs evoked in ca1 pyramidal cell synapses in hippocampal slices of glua2 mice in two distinct experimental conditions , one ensuring chelation of intracellular polyamines and the other having buffered spermine added to the intracellular solution . as in this mouse mutant all ampa channels are polyamine sensitive , we could evaluate the maximal effect of the applied concentration of phtx-433 ( 10 m ) on epscs mediated solely by glua2-lacking synaptic ampa channels . this should be useful for revealing their existence and estimating their contribution in wt , in which they would co - exist with glua2-containing ampars . additionally , we evaluated the sensitivity of synaptic ampars to intracellular polyamines in glua2 mice and compared it to that of wt . to this end , we measured the synaptic current - voltage ( i v ) relationship in ca1 pyramidal neurons dialyzed with polyamine - containing intracellular solution . evoked epscs from patched ca1 pyramidal cells of glua2 mice were recorded by stimulating schaffer collaterals at interstimulus intervals of 6 s with either polyamine - containing or -free patch - pipette solution . the washout of endogenous polyamines led to a three to fourfold increase in epsc amplitudes ( 0.29 0.27 relative to the steady - state values ; figures 1a , b ) . subsequent application of phtx-433 ( 10 m ) reduced the amplitude of responses to 0.34 0.15 compared to maximal amplitudes after polyamine washout ( n = 7 from 4 mice ; p < 0.001 ) . thus , 10 m of phtx-433 blocked 70% of the glua2-less ampa channels . however , when 0.5 mm of spermine was included in the pipette solution we typically observed a slight , though not significant , reduction in the epsc amplitude as whole - cell dialysis was progressing . initial amplitudes were 1.12 0.28 relative to the steady - state level reached after 30 min of dialysis ( p > 0.05 ; n = 7 from 5 mice ) . reduction of postsynaptic responses by subsequently applied phtx-433 was significantly less pronounced when compared to polyamine - free conditions ( 0.63 0.09 ; p < 0.01 ; figures 1a , b ) . maximal and polyamine - attenuated levels of phtx-433 blockade in ca1 pyramidal cells of glua2 mice . ( a ) the time course of epsc amplitude changes during washout of polyamine and subsequent phtx-433 ( 10 m ) application , when ca1 pyramidal neurons were dialyzed with polyamine - free intracellular solution ( green circles ) . also shown is the effect of exogenously added spermine on epsc amplitudes and the masking effect of spermine on phtx-433 suppression ( black circles ) . ( b ) representative averaged traces at the indicated times of recording in polyamine - free conditions are shown in green , in polyamine - containing condition in black . ( c ) effect of intracellular polyamines on ampar - mediated synaptic currents in glua2 and wt mice . ampar - mediated synaptic i v curves measured with polyamine - containing intracellular solution in glua2 and wt mice ( left panel ) . rectification properties of synaptic ampar channels in glua2 and wt mice ( right panel ) . ris were calculated as the ratio of epsc amplitudes measured at 30 and 70 mv ( epsc70/epsc30 ) . collectively , the data from glua2 mice suggest that endogenous or exogenously added polyamines constitutively block glua2-lacking channels and attenuate the effect of bath - applied phtx-433 . strong sensitivity of ampars to polyamines in glua2 mice was further demonstrated by measuring the synaptic i blockade in these mutants of the glua2-less ampars by polyamines resulted in a characteristic inwardly rectifying i figure 1c compares the rectification properties of ampar channels mediating synaptic transmission at schaffer collateral / commissural synapses in ca1 pyramidal cells of glua2 and wt mice . obviously , glua2 ablation leads to enhanced polyamine sensitivity relative to wt , as reflected by an increased ri ( glua2 : ri = 10.3 0.85 , n = 5 ; wt : ri = 3.9 0.77 n = 5 ; p < 0.01 ) . we next determined how the duration of agonist application affects the phtx-433 blocking potency and to what extent the current suppression by the toxin depends on intracellular polyamine content . we employed fast agonist application on outside - out patches of hek293 cells expressing homomeric glua1 receptor channels in presence of 50 m cyclothiazide to exclude an influence of channel desensitization . application of 1 mm glutamate for 100 ms at 70 mv evoked stable inward currents , which were drastically reduced in presence of 10 m phtx-433 , reaching a steady - state level of 3 1% of the initial amplitude ( figure a1a ; however , at 5 ms agonist applications , the steady - state level reached upon phtx-433 application was significantly higher ( 28 11% of control amplitudes ; figure a1b ; n = 5 ; p < 0.01 ) . similar experiments with a polyamine - free pipette solution demonstrated a reduction of glutamate - evoked currents ( 5 ms ; 1 mm ) that was stronger ( 11 7% relative to control values ; figure a1c ; n = 5 ) than the 28 11% observed with the pa - containing solution ( p < 0.05 ) . thus , usage of phtx-433 as a tool to probe the expression of glua2-lacking ampars requires the washout of endogenous polyamines . we performed similar experiments on glua1 and glua1(r ) mouse mutants , in which the likelihood for the presence in ca1 pyramidal cells of synaptic glua2-less , ca - permeable ampa channels is low and hence , sensitivity to polyamines and phtx-433 should be lacking . indeed , prolonged whole - cell dialysis with polyamine - free intracellular solution did not result in a significant increase in epsc amplitudes in ca1 pyramidal cells in either glua1 or glua1(r ) mice . we did record a moderate enhancement of ampar - mediated responses ( 0.78 0.1 and 0.81 0.11 , n = 7 cells , for 45 mice of each mutant line ) , which is most likely due to frequency facilitation or augmentation ( thomson , 2000 ; zucker and regehr , 2002 ) . as expected , application of phtx-433 ( 10 m ) did not affect epsc amplitudes in the glua1 ( 0.98 0.13 ; n = 7 from 4 mice ) or glua1(r ) mutants ( 1.07 0.16 ; n = 7 from 5 mice ) . synaptic ampar channels in ca1 pyramidal cells of glua1 and glua1(r ) mouse mutants are polyamine and phtx-433 insensitive . ( a ) the time course of epsc amplitude changes recorded in ca1 pyramidal neurons of glua1 ( green circles ) and glua1(r ) ( blue circles ) mutant mice during washout of polyamine and subsequent phtx-433 ( 10 m ) application . ( b ) representative averaged traces at the indicated times of recording from glua1 mice shown in green , and for glua1(r ) mutants in blue . ( c ) effect of intracellular polyamines on rectification properties of ampar - mediated synaptic currents in glua1 and wt animals . synaptic ampar - mediated i v curves measured with polyamine - containing intracellular solution in glua1 and wt mice ( left panel ) . rectification indices of synaptic ampar channels in glua2 and wt mice ( right panel ) . addition of 0.5 mm spermine to the intracellular solution did not cause any inward rectification of ampar - mediated synaptic currents in glua1 mice , and the i v curve became slightly outwardly rectifying ( figure 2c ) . however the shape of the i v curve and the ri measured in glua1 mice differed significantly from those in wt mice under the same experimental conditions ( figure 2c ; glua1 : ri = 2.32 0.42 , n = 5 and wt : ri = 3.9 0.77 n = 5 ; p < 0.05 ) . we conclude that absence of glua1 ( or expression of glua1 in its r - form ) greatly diminish the presence of synaptic ca - permeable ampa channels . according to previous studies , epscs recorded from ca1 pyramidal cells are insensitive to phtx-433 ( adesnik and nicoll , 2007 ; lu et al . , 2009 ) . as intracellular polyamines can occlude block by phtx-433 ( figure 1 ) , we first determined how endogenous polyamine washout changes the potency of exogenously applied phtx-433 on epsc amplitudes in wt ca1 cells . when the cells were dialyzed with polyamine - free solution , the amplitudes of evoked ampar - mediated responses increased during the first 1520 min of recording , finally reaching a steady - state level ( figures 3a , b ) . the average initial amplitude was 0.45 0.21 ( n = 9 from 6 mice ) , when normalized to the steady - state values ( 2030 min of recording ) . subsequent application of phtx-433 ( 10 m ) caused a gradual reduction of the postsynaptic responses , reaching a plateau level ( 5060 min of recording ) of 0.69 0.17 ( p < 0.01 ) , as compared to values prior to phtx-433 application ( figures 3a , b ) . ( a ) scatter plot shows epsc amplitude dynamics during prolonged whole - cell dialysis and following phtx-433 ( 10 m ) application , when ca1 pyramidal neurons were patched with either polyamine - free ( green circles ) or polyamine - containing ( black circles ) intracellular solution . ( b ) representative averaged traces at the indicated times of recording in polyamine - free ( green ) or polyamine - containing ( black ) solution . ( c ) dependence of ampar synaptic channel rectification properties in wt ca1 pyramidal cells on intracellular polyamine content . synaptic ampar - mediated i v curves measured with polyamine - containing and -free intracellular solutions ( left panel ) . dependence of rectification indices of synaptic ampar channels on the presence of polyamines in wt mice ( right panel ) . by contrast , when wt ca1 neurons were patched with polyamine - containing solution , the amplitudes of responses during the initial phase of dialysis were either stable or underwent a slight , but not significant reduction ( 1.18 0.14 , p > 0.05 , n = 7 from 5 mice ) . moreover , application of phtx-433 caused no significant changes in epsc amplitudes ( 1.12 0.14 ; p > 0.05 ) . thus , in wt ca1 pyramidal cells , washout of intracellular polyamines resulted in an increase in the amplitude of evoked postsynaptic responses , which was in large part counteracted by subsequent application of phtx-433 ( figures 3a , b ) . this remarkable difference to the results from synaptic ampars in ca1 pyramidal cells of glua1 and glua1(r ) mouse mutants constitutes unequivocal evidence for the presence of synaptic ca - permeable ampa receptors in wt ca1 cells . to further substantiate our finding on the existence of glua2-lacking ampars at schaffer collateral / commissural synapses in wt ca1 pyramidal cells , we evaluated the effect of the presence of intracellular spermine on the shape of ampar - mediated i v curves and on the ri . v relation recorded with polyamine - free solution was nearly linear and deviated strongly at positive potentials from that observed with polyamine - containing solution . also , the ri in presence of spermine was significantly higher compared to that in polyamine - free conditions [ wt ( sp ) : 3.9 0.77 , n = 5 and wt ( no sp ) : 2.86 0.28 , n = 5 ; p < 0.05 ] . the controversy concerning the presence of glua2-less , ca - permeable ampa channels in hippocampal ca1 pyramidal cell synapses ( plant et al . , 2006 ; 2010 ) prompted this study , for which we employed , in addition to wt mice , mouse mutants lacking either the major ampar subunits glua1 or glua2 or expressing the glua1(r ) subunit , which , like glua2 , drastically attenuates the ca - permeability of ampars containing it ( in preparation ) . polyamine sensitivity of glua2-lacking ampars is often used to evaluate the relative contribution of these channels to the net ampar - mediated synaptic current . endogenous or exogenously added polyamines can block these receptors , resulting in strong current rectification , which can be measured as ri . alternatively , washout of polyamines should increase epsc amplitudes in synapses in which glua2-lacking channels contribute to synaptic transmission ( rozov and burnashev , 1999 ) . the magnitude of this enhancement would depend on the endogenous polyamine concentration and the ratio between glua2-lacking and glua2-containing channels ( figures 4a , b ) . in all our experiments , we observed epsc augmentation upon polyamine washout during the initial 2025 min of recording , but the scale of this enhancement strongly depended on the particular mouse mutant . as expected , the increase was largest in glua2 mice and smallest in glua1 or glua1(r ) mutants . in the latter two mutant lines the augmentation of postsynaptic responses is unlikely to reflect the washout of polyamines , since the ampars in these mutants should be polyamine insensitive . a possible explanation for the epsc amplitude rise in these mutants might be activity dependent presynaptic phenomena , such as frequency facilitation or augmentation ( thomson , 2000 ; zucker and regehr , 2002 ) , which could also contribute to the epsc enhancement by polyamine washout in wt and glua2 mice this assumption allows us to correct the data from wt and glua2 mice and thus estimate better the putative impact of polyamine washout on fast excitatory synaptic transmission at schaffer collateral to ca1 pyramidal cell synapses ( figure 4c ) . ( a ) polyamines enter and block glua2-lacking ampars in the closed state . in glua2 mice almost all channels the channel conductance is strongly attenuated , resulting in reduced amplitudes of the net response ( middle panel ) . in contrast , washout of polyamines results in an increased ampar conductance , leading to enhanced epsc amplitudes ( right panel ) . ( b ) when glua2-lacking and glua2-containing ampars are co - expressed at the same synapse , the effect of polyamine washout will depend on the ratio between the two channel types . representative traces show enhancement of ampar - mediated epscs during polyamine washout ( wt in red , glua2 in black ) . ( c ) the plot summarizes data on the initial amplitude enhancement observed in wt , glua2 and glua1 mice . the dashed line shows the exponential fits of the time dependent amplitude augmentation during polyamine washout ( wt in red , glua2 in black and glua1 in blue ) . since the vast majority of ca1 cell ampa channels in glua1 mice should be polyamine insensitive , the slight epsc enhancement represents another intrinsic property of schaffer collateral / commisural to ca1 pyramidal cells synapses ( e.g. , frequency facilitation ) . thus , by subtracting the glua1 data from those obtained from wt or glua2 mice , we can calculate the putative effect of endogenous polyamine washout on epsc amplitudes in wt ( red line ) and glua2 ( black line ) mice . we also employed the sensitivity of synaptic ampa currents to phtx-433 to detect the presence of glua2-lacking ampars . the accuracy of this phtx-433 probing test is sensitive to intracellular polyamines . indeed , as we show , the presence of 40 m of free spermine reduces the phtx-433 suppression of ampar - mediated epscs by 50% in glua2 mice , in which all ampa channels should be sensitive to the wasp toxin . the use of glua2 mice allowed us to determine that after polyamine washout , 10 m phtx-433 blocks 70% of the epscs mediated by glua2-lacking ampa channels . the epscs mediated by ampa channels in glua1 or glua1(r ) mutants were insensitive to phtx-433 , consonant with the predicted lack of ca - permeable ampa channels in these mutant mice , and thus demonstrating that 10 m phtx-433 exerts no unspecific effects on synaptic transmission . notably , following polyamine washout , 10 m phtx-433 blocked 31% of the epscs in wt ca1 pyramidal cell synapses , clearly demonstrating the presence of ca - permeable glua2-less ampa channels . while in glua2 mice , where all ampars are polyamine sensitive , phtx blocked 66% of the epsc . by solving a simple rule of proportion we can calculate that in wt ca1 pyramidal cells , under artificial polyamine - free conditions , glua2-lacking channels mediate 47% of the net epsc . to estimate the proportion of such channels in the entire synaptic ampar population , one needs to consider that the conductance of glua2-lacking ampars is 510 times higher than that of glua2-containing channels . these data allow us to estimate the relative contribution of glua2-lacking channels to synaptic transmission at schaffer collateral ca1 cell synapses . in polyamine - free conditions , recombinant glua2-lacking channels have a maximal conductance of 1925.5 ps , whereas the conductance of glua2-containing ampars is 2.5 ps ( oh and derkach , 2005 ) . we thus estimate the population of glua2-lacking ampars to be in the range of 810% . under native conditions , the contribution of glua2-lacking channels to the synaptic response depends on the intracellular polyamine concentration and should , therefore , be < 47% . however , during repetitive activity , the impact of these channels will increase due to the activity - dependent relief from polyamine block . also , the degree of the block could be significantly attenuated by interaction of glua2-lacking ampars with tarps . thus , the role of polyamine - sensitive ampars at schaffer collateral / commissural synapses might be differentially regulated during shorter and longer activity epochs , thus providing additional ways to control the synaptic gain . our analyses might resolve the apparent disagreement in the field regarding glua2-lacking ampars in ca1 pyramidal cells . the phtx-433 test becomes less sensitive in the presence of intracellular polyamines , since the toxin can only block channels , which are not occupied by internal polyamines . crucially , polyamines block glua2-lacking channels in the closed state , and the block itself is not voltage dependent . therefore , to maximize the measurable effect of phtx-433 , the washout of endogenous polyamines becomes essential , lest the contribution of these channels to synaptic transmission is underestimated , as demonstrated here by fast agonist application experiments on recombinant glua1 homomeric ampars . the presence of ca - permeable ampa channels in ca1 cell synapses prompts the question as to their functional role . plant et al . ( 2006 ) observed transient incorporation of such ampa channels following induction of hippocampal ltp and reported that the transient ca - influx is essential for the subsequent maintenance phase of ltp . indeed , the contribution of the ca - influx via gated ca - permeable ampa channels is very low relative to that of nmda channels , considering an approximately 10-fold lower fractional ca - current and a > 10-fold lesser envelope of charge transfer of the ampa channels . notably , our data indicate no need for the transient incorporation of ca - permeable ampa channels into ca1 cell synapses upon ltp induction ( plant et al . , 2006 ) , given that such channels already constitute a small subpopulation of the synaptic ampa receptors in wt ca1 pyramidal neurons . rather , it appears that ltp induction may facilitate the participation in excitatory transmission of the synaptic ca - permeable ampa channels , conceivably . via phosphorylation of intracellular subunit domains , with the additional negative charges close to the inner channel mouth accelerating the unblocking of polyamines . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

[ purpose ] the purpose of this study was to determine the normal range of the side - to - side difference in three dimensional knee kinematics measured by the point cluster technique ( pct ) . [ subjects ] the subjects were twenty - one healthy normal volunteers without knee pain or an episode of injury to the legs . [ methods ] the subjects were tested bilaterally at a self - selected normal walking speed and six degrees of freedom knee kinematics were measured using the pct , and the 95% confidence intervals of the average side - to - side differences in flexion - extension ( fe ) , adduction - abduction ( aa ) , internal - external ( ie ) rotation , and anterior - posterior ( ap ) , medial - lateral ( ml ) , superior - inferior ( si ) translation in each stage of the gait cycle were determined . [ results ] the average side - to - side differences and their 95% confidence intervals in rotation / translation in each stage of the gait cycle were determined . the side - to - side differences in aa rotation and ap translation of the tibia were significantly larger in the swing phase than in the stance phase . [ conclusion ] the side - to - side differences in aa rotation and ap translation were highly dependent on the stage of the gait cycle . therefore , the normal ranges of the side - to - side differences in knee kinematics in each stage of the gait cycle , in particular aa rotation and ap translation of the tibia , is useful information for evaluating knee kinematics during walking . the glua2 subunit in heteromeric alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) receptor channels restricts ca2 + permeability and block by polyamines , rendering linear the current - voltage relationship of these glutamate - gated cation channels . although glua2-lacking synaptic ampa receptors occur in gaba - ergic inhibitory neurons , hippocampal ca1 pyramidal cell synapses are widely held to feature only glua2 containing ampa receptors . a controversy has arisen from reports of glua2-lacking ampa receptors at hippocampal ca3-to - ca1 cell synapses and a study contesting these findings . here we sought independent evidence for the presence of glua2-lacking ampa receptors in ca1 pyramidal cell synapses by probing the sensitivity of their gated cation channels in wild - type ( wt ) mice and gene - targeted mouse mutants to philanthotoxin , a specific blocker of glua2-lacking ampa receptors . the mutants either lacked glua2 for maximal philanthotoxin sensitivity , or , for minimal sensitivity , expressed glua1 solely in a q / r site - edited version or not at all . our comparative electrophysiological analyses provide incontrovertible evidence for the presence in wild - type ca1 pyramidal cell synapses of glua2-less ampa receptor channels . this article is part of a special issue entitled calcium permeable ampars in synaptic plasticity and disease .

lignocellulosic materials represent an abundant and inexpensive source of sugars and can be microbiologically converted to industrial products . xylitol ( c5h12o5 ) , a sugar alcohol obtained from xylose , is generated during the metabolism of carbohydrates in animals and humans . xylitol was present in fruits and vegetables , at low concentration , which makes its production from these sources economically unfeasible . its sweetening power was comparable to that of sucrose and is higher than that of sorbitol and mannitol . in addition to reducing dental caries , xylitol also promotes tooth enamel remineralization by reversing small lesions . this happens because , when in contact with xylitol , the saliva seems to be favorably influenced ; the chemical composition of xylitol induces the calcium ions and phosphate . for these characteristics , xylitol was a feed stock of great interest to food , odontological , and pharmaceutical industries . however it was expensive and it requires several steps of xylose purification before the chemical reaction [ 4 , 9 , 10 ] . xylitol production through bioconversion has been proposed as for utilizing microorganism such as yeast , bacteria , and fungi [ 11 , 12 ] . among these , yeast has shown to possess desirable properties for xylitol production [ 13 , 14 ] . therefore , for the present study , yeast strain pachysolen tannophilus was selected for xylitol production . furthermore studies have shown that nutritional factors including sources of carbon and nitrogen can influence xylitol production . corncob is a large volume solid waste for using sweet corn processing industry in india . they are currently used as animal feed or returned to the harvested field for land application . corncob contains approximately over 40% of the dry matter in residues and thus has value has a raw material for production of xylose , xylitol , arabinose , xylobiose , and xylo oligosaccharides . these carbohydrates mainly consist of the xylose and other minor pentose [ 1820 ] . among various agricultural wastes , corncob was regarded as promising agricultural resources for microbial xylitol production . in microbial production of xylitol from corncob , the cobs were first hydrolysed to produce from hemicelluloses by acid hydrolysis and the corncob hydrolysate is then used as the medium for xylitol production . the bioconversion of xylitol is influenced by the concentration of various ingredients in culture medium . this study also investigates the effect of process variables such as ph , temperature , substrate concentration , inoculum size , and agitation speed on xylitol yield . response surface methodology ( rsm ) is a mathematical and statistical analysis , which is useful for the modeling and analysis problems that the response of interest is influenced by several variables . rsm was utilized extensively for optimizing different biotechnological process [ 22 , 23 ] . in the present study , the screening and optimization of medium composition and process variables for xylitol production by pachysolen tannophilus using plackett - burman and rsm were reported . the plackett - burman screening design was applied for knowing the most significant nutrients enhancing xylitol production . then , box - behnken design and central composite design ( ccd ) were applied to determine the optimum level of significant nutrients and process variables , respectively . the yeast strain pachysolen tannophilus ( mtcc 1077 ) was collected from microbial type culture collection and gene bank , chandigarh . the lyophilized stock cultures were maintained at 4c on culture medium supplemented with 20 g of agar . the medium composition ( g / l ) was compressed of the following : malt extract : 3.0 ; yeast extract : 3.0 ; peptone : 5.0 ; glucose : 10.0 at ph : 7 . corncob was collected from perambalur farms , tamil nadu , india , and was dried in sunlight for 2 days , crushed , and sieved for different mesh size ranging from 0.45 mm to 0.9 mm ( 2040 mesh ) and used for further studies . the composition of the corncob ( g / l ) : xylose : 28.7 , glucose : 5.4 , arabinose : 3.7 , cellobiose : 0.5 , galactose : 0.7 , mannose : 0.4 , acetic acid : 2 , furfural : 0.8 , hydroxymethyl furfural : 0.2 was used for xylitol production . 2 g of corncobs at a solid loading of 10% ( w / w ) was mixed with dilute sulfuric acid ( 0.1% ( w / v ) ) and pretreated in an autoclave at 120c with residence time of 1 hour . the liquid fraction was separated by filtration and the unhydrolysed solid residue was washed with warm water ( 60c ) . hemicellulose acid hydrolysate was heated at 100c for 15 min to reduce the volatile components . the hydrolysate was overlined with solid ca(oh)2 up to ph 10 , in combination with 0.1% sodium sulfite , and filtered to remove the insoluble materials . activated charcoal treatment was an efficient and economic method of reduction in the amount of phenolic compounds , acetic acid , aromatic compounds , furfural , and hydroxymethylfurfural normally found in hemicellulosic hydrolysates . after centrifugation , the solutions were mixed with powdered charcoal at 5% ( w / v ) for 30 and stirred ( 100 rpm ) at 30c . the liquor was recovered by filtration , chemically characterized , and used for culture media . fermentation was carried out in 250 ml erlenmeyer flasks with 100 ml of pretreated corncob hemicelluloses hydrolysate is adjusted to ph 7 with 2 m h2so4 or 3 m naoh and supplemented with different nutrients concentration for tests according to the selected factorial design , were used for fermentation medium and sterilized at 120c for 20 mins . the flasks were maintained at 30c for agitation at 200 rpm for 48 hours . after the optimization of medium composition , the fermentation was carried out with different parameter levels ( table 5 ) with the optimized media for tests according to the selected factorial design . during the preliminary screening , the experiments were carried out for 5 days and the maximum production was obtained in 48 hours . sugar and sugar alcohol in the culture broth were measured by high - performance liquid chromatography ( hplc ) , model lc-10-ad ( shimadzu , tokyo , japan ) equipped with a refractive index ( ri ) detector . the chromatography column used was a aminex hpx-87h ( 300 7.8 mm ) column at 80c with 5 mm h2so4 as mobile phase at a flow rate of 0.4 ml / min , and the injected sample volume was 20 l . the rsm has several classes of designs , with its own properties and characteristics . central composite design ( ccd ) , box - behnken design , and three - level factorial design are the most popular designs applied by the researchers . a prior knowledge with understanding of the related bioprocesses is necessary for a realistic modeling approach . it assumes that there are no interactions between the different variables in the range under consideration . plackett - burman experimental design is a fractional factorial design and the main effects of such a design may be simply calculated as the difference between the average of measurements made at the high level ( + 1 ) of the factor and the average of measurements at the low level ( 1 ) . to determine which variables significantly affect xylitol production , nine variables were screened in 12 experimental runs ( table 1 ) , and insignificant ones are eliminated in order to obtain a smaller , manageable set of factors . the low level ( 1 ) and high level ( + 1 ) of each factor ( 1 , + 1 ) g / l ) : k2hpo4 ( 6.6 , 7 ) , yeast extract ( 1.5 , 5 ) , peptone ( 2 , 5 ) , kh2po4 ( 1.2 , 3.6 ) , xylose ( 9.8 , 10.2 ) , ( nh4)2so4 ( 1 , 4 ) , mgso47h2o ( 0.7 , 1.3 ) , malt ( 2.8 , 3.2 ) , and glucose ( 9.8 , 10.2 ) , and they were coded with a , b , c , d , e , f , g , h , i , respectively . the statistical software package minitab 16 is used for analyzing the experimental data . once the critical factors are identified through the screening , the box - behnken design was used to obtain a quadratic model after the central composite design ( ccd ) was used to optimize the process variables and obtain a quadratic model . the box - behnken design and ccd was used to study the effects of the variables towards their responses and subsequently in the optimization studies . this method was suitable for fitting a quadratic surface , and it helps to optimize the effective parameters with a minimum number of experiments , as well as to analyze the interaction between the parameters . in order to determine the relationship between the factors and response variables , a regression design was employed to model a response as a mathematical function ( either known or empirical ) for few continuous factors , and good model parameter estimates are desired . the coded values of the process parameters are determined by the following equation : ( 1)xi = xix0x , where xi is coded value of the ith variable , xi is uncoded value of the ith test variable , and x0 is uncoded value of the ith test variable at center point . the regression analysis is performed to estimate the response function as a second - order polynomial : ( 2)y=0+i=1kixi+i=1kiixi2i=1 , i < jk1 j=2kijxixj , where y is the predicted response , 0 constant , and i , ii , ij are coefficients estimated from regression . they represent the linear , quadratic , and cross - products of xi and xj on response . the regression and graphical analysis are carried out using design - expert software ( version 7.1.5 , stat - ease , inc . , the adequacy of the models is further justified through analysis of variance ( anova ) . lack - of - fit is a special diagnostic test for adequacy of a model that compares the pure error , based on the replicate measurements to the other lack of fit , based on the model performance . f value , calculated as the ratio between the lack - of - fit mean square and the pure error mean square , is the statistic parameter used to determine whether the lack - of - fit is significant or not , at a significance level . the statistical model was validated with respect to xylitol production under the conditions predicted by the model in shake - flask level . samples were drawn at the desired intervals and xylitol production was determined as described above . plackett - burman experiments ( table 1 ) showed a wide variation in xylitol production . this variation reflected the importance of optimization to attain higher productivity . from the pareto chart shown in figure 1 the variables , namely , peptone , xylose , mgso47h2o , and yeast extract , were selected for further optimization to attain a maximum response . the levels of factors and the effect of their interactions on xylitol production were determined by box - behnken design of rsm . the design matrix of experimental results by tests was planned according to the 29 full factorial designs . twenty - nine experiments were performed at different combinations of the factors shown in table 2 , and the central point was repeated five times . the predicted and observed responses along with design matrix are presented in table 3 , and the results were analyzed by anova . the second - order regression equation provided the levels of xylitol production as a function of peptone , xylose , mgso47h2o , and yeast extract , which can be presented in terms of coded factors as in the following equation : ( 3)y=0.70 + 0.053a+0.018b+0.057c+0.054d + 0.092ab(2.500e003)ac + ( 1.000e002)ad+0.028bc+0.077bd 0.040cd0.083a20.16b20.076c20.11d2 , where y is the xylitol yield ( g / g ) and a , b , c , and d were peptone , xylose , mgso47h2o , and yeast extract , respectively . there is only a 0.01% chance that a model f - value this large could occur due to noise . values of prob > f less than 0.05 indicate that model terms are significant . values greater than 0.1 indicate that model terms are not significant . in the present work , linear terms of a , c , and d and all the square effects of a , b , c , and d and the combination of ab , bd , and cd were significant for xylitol production . the coefficient of determination ( r ) for xylitol production was calculated as 0.9634 , which is very close to 1 and can explain up to 96.00% variability of the response . the predicted r value of 0.7898 was in reasonable agreement with the adjusted r value of 0.9267 . the adequate precision value of 16.010 indicates an adequate signal and suggests that the model can navigate the design space . the above model can be used to predict the xylitol production within the limits of the experimental factors that the actual response values agree well with the predicted response values . experimental conditions for optimization of the process variables for xylitol yield were determined by ccd of rsm . the design matrix of experimental results by tests was planned according to the 50 full factorial designs , and the central point was repeated eight times . the predicted and observed responses along with design matrix are presented in table 6 and the results were analyzed by anova . the second - order regression equation provided the levels of xylitol production as a function of temperature , substrate concentration , ph , agitation speed , and inoculums size , which can be presented in terms of coded factors as in the following equation : ( 4)y=0.79 + 0.025a+0.043b+0.049c + 0.030d+0.038e0.029ab(4.063e003)ac + 0.018ad+(2.187e003)ae(9.688e003)bc + 0.014bd+(5.312e003)be+(1.562e003)cd + ( 5.312e003)ce(3.125e004)de0.040a2 0.041b20.046c20.027d20.034e2 , where y was the xylitol yield ( g / g ) and a , b , c , d , and e are temperature , substrate concentration , ph , agitation speed , and inoculums size , respectively . there is only a 0.01% chance that a model f - value this large could occur due to noise . values of prob > f less than 0.05 indicate that model terms are significant . values greater than 0.1 indicate that model terms are not significant . in the present work , linear terms and all the square effects of a , b , c , d , and e and the coefficient of determination ( r ) for xylitol production was calculated as 0.9148 , which is very close to 1 and can explain up to 91.00% variability of the response . the predicted r value of 0.6867 was in reasonable agreement with the adjusted r value of 0.8561 . the adequate precision value of 12.951 indicates an adequate signal and suggests that the model can navigate the design space . in both designs the interaction effects of variables on xylitol production were studied by plotting 3d surface curves against any two independent variables , while keeping another variable at its central ( 0 ) level . the 3d curves of the calculated response ( xylitol yield ) and contour plots from the interactions between the variables were obtained . this evidence from above figures shows the dependency of xylose , mgso47h2o , yeast extract on xylitol production . the optimal operation conditions of peptone , xylose , mgso47h2o , and yeast extract for maximum xylitol production were determined by response surface analysis and also estimated by regression equation . the predicted values from the regression equation closely agreed with that obtained from experimental values . the xylitol production increased with increase in temperature to about 36c , and thereafter xylitol production decreased with further increase in temperature . this evidance from above figures shows the dependency of ph , substrate concentration , agitation speed , and inoculum size on xylitol production . the optimal operation conditions of temperature , substrate concentration , ph , agitation speed , and inoculum size for maximum xylitol production were determined by response surface analysis and also estimated by regression equation . the predicted values from the regression equation closely agreed with that obtained from experimental values . validation of the experimental model was tested by carrying out the batch experiment under optimal operation conditions which are ( g / l ) : peptone : 6.03 , xylose : 10.62 , mgso47h2o : 1.39 , yeast extract : 4.66 established by the regression model . under optimal process variables levels are temperature ( 36.56c ) , ph ( 7.27 ) , substrate concentration ( 3.55 g / l ) , inoculum size ( 3.69 ml ) , and agitation speed ( 194.44 rpm ) . the xylitol production ( 0.80 g / g ) obtained from experiments was very close to the actual response ( 0.78 g / g ) predicted by the regression model , which proved the validity of the model . in this work , plackett - burman design was used to test the relative importance of medium components on xylitol production . among the variables , peptone , xylose , mgso47h2o , and yeast g / l ) : peptone : 6.03 , xylose : 10.62 , mgso47h2o : 1.39 , and yeast extract : 4.66 . then the influence of various process variables , namely , temperature , ph , substrate concentration , agitation speed , and inoculum size on the xylitol production was evaluated by ccd . the optimum levels of process variables are temperature ( 36.56c ) , ph ( 7.27 ) , substrate concentration ( 3.55 g / l ) , inoculum size ( 3.69 ml ) , and agitation speed ( 194.44 rpm ) . this study showed that the corncob is a good source for the production of xylitol . using the optimized conditions , the xylitol yield reaches 0.80 g / g . osteoporosis is a systematic bone disease characterized by low bone mass and deterioration of microarchitecture of the bone , leading to bone fragility and eventually fractures 1 . the gold standard method recommended by the world health organization in the diagnosis of osteoporosis is dual - x - ray absorptiometry ( dxa ) . using this method , osteoporosis is defined by a bone mineral density ( bmd ) lower than -2.5 standard deviations ( sd ) of the reference bmd of caucasian women aged 20 - 29 years 2 . undeniably , this simplified definition of osteoporosis eases the physicians in diagnosing and initiating treatment for osteoporotic patients . however , there are several limitations of dxa which prevent it from being used in the mass screening of osteoporosis , which is currently a rising healthcare medical condition in the developing countries 3 . quantitative ultrasound ( qus ) is a bone health assessment technique which has gained much popularity in recent years since its introduction in 1984 . compared to dxa , qus offers wider accessibility to the public because it is portable , easier to handle , lower in cost and does not emit ionizing radiation 4 . this technology has been used to determine the bone health status in women 5 , men 6 , children 7 and in certain cases , infants 8 . the ultrasound is a type of sound wave with a frequency exceeding the normal auditory range of humans ( > 20 khz ) . the sound waves produced by unique piezoelectric probes are emitted and travelled longitudinally or horizontally through the bone under study . there are usually two probes on the qus device : the emission and receiver probes . the segment of bone under study will be placed between these probes and the ultrasound waves emitted from the emission probes through the bone will be sensed by the receiver probe 9 . there are two types of qus depending on the axis the ultrasound waves take to travel through the bone . horizontal transmission uses probes that measure the speed of sound on the cortical layer of the bone at a fixed distance . longitudinal transmission is more often used and the bone segment measured is the calcaneus 4 . according to the international society of clinical densitometry ( iscd ) , calcaneal qus is the only recognized measurement of qus as the determinant of bone health status because more research has been performed on the calcaneus as compared to the other bone segments 10 . besides , the calcaneus consists of 95% trabecular bone and possesses two lateral surfaces 11 , which facilitates the movement of ultrasound through it . therefore , this discussion will emphasize on this measurement technique , which is the calcaneal qus . two parameters commonly generated by qus are the speed of sound ( sos ) and the broadband ultrasound attenuation ( bua ) . the speed of sound refers to the division of transmission time of the sound waves by the length of the body part studied . the unit used in the measurement of sos is meter per second ( m / s ) . broadband attenuation of sound refers to the slope between attenuation of sound signals and its frequency , and the unit used is db / mhz . attenuation occurs because the energy is absorbed by the soft tissue and bone when the sound waves travel through them . currently , more sophisticated qus indices derived from these two basic measurements are available , such as amplitude - depend sos ( ad - sos ) , stiffness index ( si ) , quantitative ultrasound index ( qui ) and estimated bmd ( ebmd ) . some researchers suggested that these composite parameters are more useful in the determination of subjects with low bone health status 9 . previous in vitro studies examining the relationship between calcaneal qus and bone properties found that sos was closely related to bmd 12 - 16 . toyras et al . indicated that this relationship was strong , with a coefficient of correlation ( r ) of 0.888 17 . significant correlations between sos with microarchitecture indices of the bone , such as bone volume ( bv / tv ) , bone surface ( bs / tv ) , number of nodes ( n.nd . ) , trabecular number ( tb.n . ) , trabecular thickness ( tb.th . ) and trabecular separation ( tb.sp . ) there were opinions that these correlations were mediated by the bone mass , and if bmd was controlled , these relationships would revert to become non - significant 18 . however , a computer simulation study performed by haat et al . showed that after adjusting for bmd , bv / tv remained significantly associated with sos 20 . this was confirmed by later studies using excised samples , whereby microarchitecture of the bone was significantly associated with sos and contributed to the variation of sos apart from bmd 12 , 15 . bone biomechanical studies revealed that young 's modulus , compressive modulus , ultimate strength and elasticity of bone were significantly associated with sos 12 , 16 , 21 . indicated that the combination of bone density and young 's modulus could explain 93.34% of the in vitro variation of sos 12 . studies also showed that bua was significantly associated with biomechanical parameters 21 , but toyras et al . indicated that this was only true in low - density bone samples 16 , 17 . in high - density bovine samples , these in vitro experiments ( summarized in table 1 ) showed that qus indices are able to reflect the two principal constituents of bone health , which are the bone quantity ( bmd and bone mass ) and the bone quality ( bone microarchitecture and strength ) . the stronger association between qus indices and bmd indicates that bone quantity contributes to most of the variation in qus 22 . in humans , there were significant correlations between qus indices and bmd values at various body sites assessed cross - sectionally . reported that all three qus indices , bua , sos and si were significantly correlated to bmd at lumbar spine and femur in postmenopausal women , but only sos correlated significantly to bmd at lumbar spine and femur in premenopausal women 23 . in a study by mszros et al . in men , bua correlated significantly and moderately with bmd at lumbar spine , femoral neck and radius midshaft . however , sos did not correlate with the bmd at the aforementioned sites 6 . in a longitudinal study by trimpau et al . involving 80 swedish women aged 53 - 73 years , bua and sos were significantly correlated with bmd at multiple skeletal sites at the first screening and the after seven years later . furthermore , the changes of dxa and qus measurements during the follow - up period were also significantly correlated 24 . the ability of qus to predict fractures were also validated in several human cohort studies . hernandez et al . examined 5195 spanish postmenopausal women 65 years and found that all qus indices ( bua , sos , ebmd and qui ) were significantly different between subjects with and without history of fractures . logistic regression analysis also confirmed that these qus indices were significantly associated with previous fractures 25 . the study of varenna et al . in 4832 italian men aged 60 - 80 years found that qus indices ( bua , sos , si ) were significant associated with history of hip fracture and non - spinal fracture 26 . these observations from cross - sectional studies were further validated by prospective studies . in the norfolk cohort study involving 14824 men and women discovered that one sd decrease in ultrasound velocity translated to a 60% increase in fracture risk in both genders . they also found that the risk increased for older subjects and doubled for subjects with history of fractures 27 . in the asian population , fujiwara et al . showed that sos , bua and si significantly predicted hip , wrist and non - spinal fractures in japanese men and women followed for 5 years 28 . in a recent meta - analysis , moayyeri et al . concluded that sos , bua , si and qui significantly predicted fractures after reviewing 21 independent studies 29 ( table 2 ) . several studies showed that the fracture prediction by qus was equal and sometimes better than dxa . in cross - sectional studies by mszros et al . 6 and gonnelli et al.30 involving male subjects , both studies also revealed that sos had better or the same discriminatory ability than bmd 6 , 30 . in a longitudinal study by chan et al . , the combination of bua and femoral neck bmd predicted hip , vertebral or any fractures better than individual indices in postmenopausal women but not in men followed for 13 years 31 . on the other hand , some studies reported that bmd had better discriminability than qus , and the combination of dxa and qus did not improve predictability . hong kong chinese men followed for 6.5 years demonstrated that bua , qui and bmd ( hip and spine ) significantly predicted major fragility fractures and non - vertebral fractures , but bmd was better in predicting the former . in addition , the combination of bmd and qus did not improve fracture predictability 32 . reported that only lumbar spine bmd predicted vertebral fractures in postmenopausal women but qus did not 33 . . showed similar results in pre- and postmenopausal women in their study 23 . in view of the heterogeneity of the results on the comparison , it was revealed that the predictability of qus in non - spinal fractures was similar to dxa , but dxa was more superior in predicting hip fractures 34 ( table 2 ) . according to the official position of iscd on qus bone assessment , several aspects concerning the use of t - score , reference range , precision and inter - device comparison were addressed 10 . in the classification of low bone density using dxa , t - score with cut - off points of -1.0 sd for osteopenia and -2.5 sd for osteoporosis however , the use of the same cut - off points in qus measurement is not recommended because qus and dxa essentially employ different technology in assessing bone health 10 . several studies also showed that simply applying the conventional dxa cut - offs in qus measurement significantly underestimates the true prevalence of osteoporosis 35 , 36 . a number of cut - off points for bone health classifications for qus had been suggested previously , but they were specific to the device used 37 - 39 . as an example , frost et al . reported that t - score cut off values for osteoporosis were -1.61 , -1.94 and -1.90 for bua , sos and ebmd measured using hologic sahara ultrasonometer , and -1.45 and -2.10 for bua and sos measured using osteometer dtuone 38 . the use of an appropriate reference range is important for accurate classification of bone health using qus and dxa . for example , a caucasian reference range , where bone density outcomes are typically higher than asian bone density outcomes , will eventuate in asian subjects being classified as having low bone density . chin et al . reported that even the use of references from different asian countries caused significant discrepancies in the classification of subjects with low bone health 40 . the normative values for different populations around the world had been generated for various qus devices 36 , 40 - 44 . both the population of interest and the device used should be considered when incorporating the respective normative values in qus device . due to the fact that numerous qus devices have been developed by many manufactures , each with its own designed logarithm for the calculation and interpretation of qus indices , inter - device comparison of the results of bone health assessment the precision of qus devices was reported to be poorer compared to dxa devices 45 . this may be one of the reason qus devices are not recommended for patient follow - up in the treatment of osteoporosis unless dxa is inaccessible 10 . the precision values of sos and bua are different due to the effect of a large denominator of the former , hence sos tends to have smaller precision values 45 . an example for precision values reported for the cuba mccue instrument were 2.4% for bua and 0.3% for sos 46 . we suggest that for the use of qus in the screening of bone health in local community , an ultrasonometer validated against dxa should be used . the ultrasonometer should also be equipped with the local reference curve ( or a reference curve from a population with the most similar background ) for the purpose of bone health classification . short - term and long - term in vivo precision of the device should be established for the purpose of the follow - up of subjects . furthermore , qus results should be interpreted with clinical risk factors for maximal detection of subjects with osteoporosis . several studies also indicated that it can predict fractures for both genders . in developing countries with poor accessibility to dxa , qus could be used as an effective screening tool for early detection of osteoporosis . an early detection would allow preventive measures to be taken to hinder the progression of the osteoporosis .

optimization of the culture medium and process variables for xylitol production using corncob hemicellulose hydrolysate by pachysolen tannophilus ( mttc 1077 ) was performed with statistical methodology based on experimental designs . the screening of nine nutrients for their influence on xylitol production was achieved using a plackett - burman design . peptone , xylose , mgso47h2o , and yeast extract were selected based on their positive influence on xylitol production . the selected components were optimized with box - behnken design using response surface methodology ( rsm ) . the optimum levels ( g / l ) were peptone : 6.03 , xylose : 10.62 , mgso47h2o : 1.39 , yeast extract : 4.66 . the influence of various process variables on the xylitol production was evaluated . the optimal levels of these variables were quantified by the central composite design using rsm , for establishment of a significant mathematical model with a coefficient determination of r2 = 0.91 . the validation experimental was consistent with the prediction model . the optimum levels of process variables were temperature ( 36.56c ) , ph ( 7.27 ) , substrate concentration ( 3.55 g / l ) , inoculum size ( 3.69 ml ) , and agitation speed ( 194.44 rpm ) . these conditions were validated experimentally which revealed an enhanced xylitol yield of 0.80 g / g . quantitative ultrasound ( qus ) has emerged as a convenient and popular screening tool for osteoporosis . this review aimed to provide basic information on the principle of qus measurement and discuss the properties of bone reflected by qus indices . qus employed high frequency sound waves generated by the device to determine bone health status in humans . in vitro studies showed that qus indices were significantly associated with bone mineral density ( bmd ) , bone microarchitecture and mechanical parameters . in humans , qus indices were found to be associated with bmd as well . in addition , qus could discriminate subjects with and without fracture history and predict risk for future fracture . in conclusion , qus is able to reflect bone quality and should be used in the screening of osteoporosis , especially in developing countries where dual - x - ray absorptiometry devices are less accessible to the general population .

thrombocytosis is commonly seen in clinical practice and often the cases reported are incidental . the differential diagnosis for thrombocytosis is broad and the diagnostic process can be challenging . in general , causes of thrombocytosis can be described as spurious , reactive , or clonal in nature . a number of population studies have examined the degree of thrombocytosis as well as the frequency of various etiologies of thrombocytosis when it occurs . reactive causes are by far the most common etiology of thrombocytosis in these population studies , comprising 8897% of cases in adults in two large case series and 100% of pediatric cases in a single case series . extreme thrombocytosis , defined as a platelet count > 1,000 10/l is quite rare , as only 25.8% of patients demonstrate this degree of thrombocytosis upon presentation . although often thought to be more common in clonal processes , extreme thrombocytosis can also be due to reactive causes , with 82% of cases of extreme thrombocytosis in one series being reactive in nature . a 26-year - old male patient was scheduled for surgery of right indirect inguinal hernia with right sided encysted hydrocele of the cord . the preoperative laboratory tests reported normal coagulation and biochemical parameters , haemoglobin values of 15.1 g% , no leukocytosis and a platelet count of 2.60 lakhs / cumm . patient was operated , right sided mesh hernioplasty with excision of hydrocele of the cord was done . there were no adverse incidents during surgery , the patient was smoothly extubated in the surgery room and transferred to the post anaesthesia care unit . a routine postoperative analytical blood control was performed , showing high platelets count ( 1,100,000/cumm ) , hemoglobin 11.6 mg / dl and leukocytes 18,400/cumm , with normal values of coagulation and biochemistry tests . another blood analysis was extracted to verify these data , which again returned a high platelet count ( 1,300,000/cumm ) . we suggested monitoring platelet count throughout the postoperative period by serial analytical , with introduction of thromboembolic prophylaxis and control of bleeding in the face of the probability of some degree of platelet dysfunction . the immediate period after surgery was uneventful and the patient was discharged to the ward with hemodynamic stability . later , on the next postoperative day , patient developed huge scrotal haematoma with ecchymosis around the incision line . scrotal haematoma was explored , clots were drained , but no obvious bleeder could be identified . postoperative antithrombotic prophylaxis and bleeding monitoring were maintained until the patient was discharged after 2 weeks of admission , to follow up with outpatient treatment . analytical blood controls showed that the platelet count was down to normal in 20 days . thrombocytosis is an incidental finding in 3550% of cases and determination of the cause creates a diagnostic challenge . the causes of thrombocytosis can be described as spurious , reactive , or clonal in nature as shown in table 1 . causes of thrombocytosis the reactive thrombocytosis , also called secondary thrombocytosis is the most common type and appears after acute inflammatory , infectious , neoplastic and stress processes . in these scenarios the levels of thrombopoietin , interleukin-6 and catecholamines are very high , and are thought to be responsible for the increased number of platelets . it is always necessary to distinguish between clonal ( also known as primary or essential thrombocytosis ) and reactive ( or secondary ) thrombocytosis , because their treatment and prognosis differ . distinguishing clinical features for primary and secondary thrombocytosis although the diagnostic tests to differentiate essential and reactive thrombocytosis are not easy to perform , laboratory tests that show increased acute phase reactants such as c - reactive protein , fibrinogen , erythrocyte sedimentation rate and interleukin-6 may be useful in the diagnosis of reactive thrombocytosis . it is accepted that upto 1,000,000 l platelets level are a benign condition , although it remains unclear if these findings are associated with an increased postoperative thromboembolic or haemorrhagic risk . prophylactic treatment with platelet inhibitors in these situations is controversial , although some authors do consider management of low - dose acetylsalicylic acid appropriate . reactive thrombocytosis is generally felt thought to be a self - limited process which resolves with resolution of the underlying disorder when possible . the risk of thrombotic complications with reactive thrombocytosis is felt to be low , as 1.6% of patients with reactive thrombocytosis had thrombotic complications in one large case series . all of these thrombotic events were venous in location and occurred in patients with other risk factors ( postoperative setting or underlying malignancy ) . even in cases of extreme reactive thrombocytosis the appearance of thrombocytosis after surgery needs to be diagnosed to establish the type of thrombocytosis ( clonal or reactive ) , as treatment and prognosis are quite different between them . and at all times , the surgeon must remain vigilant due to the possible risk of bleeding or thromboembolic complications . the work described in this paper was supported jointly by department of pathology and department of surgery , himsr , new delhi . the authors would like to thank hamdard institute of medical sciences for the administrative support . the authors also thank the editor and the reviewers for their valuable comments and suggestions that have led to the substantial improvement of the paper . the work described in this paper was supported jointly by department of pathology and department of surgery , himsr , new delhi . the authors would like to thank hamdard institute of medical sciences for the administrative support . the authors also thank the editor and the reviewers for their valuable comments and suggestions that have led to the substantial improvement of the paper . dementia is defined as a decline in cognitive function that is associated with an impaired function in daily life.1 the most common form of dementia is alzheimer s disease ( ad ) , which represents about 70% of cases.2 ad can be identified as a gradually progressive dementia of insidious onset , which occurs in the absence of other diseases that could be responsible for the cognitive deficits . although certain diagnostic criteria emphasize that ad is a diagnosis of exclusion with regard to vascular disease , vascular factors may also have a role in late - onset ad.1,2 previous studies suggest that there is a close relationship between ad , hypertension , diabetes , stroke , and atherosclerosis.37 some studies have investigated the parameters of arterial stiffness in patients with dementia.811 it has been observed that patients with ad have structural and functional heart abnormalities , including subclinical impairment of cardiac structure and function , aortic valve thickening , aortic regurgitation , and left ventricular ( lv ) wall motion abnormalities.12 in this study , our objective was to examine the lv diastolic parameters , atrial conduction times , and aortic stiffness parameters by conventional echocardiography in patients with and without ad . patients attending the haydarpasa numune hospital neurology clinic , istanbul , turkey , were asked to volunteer for the study if they fulfilled the inclusion criteria for ad . the control group included age- and sex - matched individuals who had no cognitive decline . all patients and their carers read the information sheet provided before giving verbal and written consent to participate in the study . the echocardiographic examinations were performed in siyami ersek cardiovascular and thoracic surgery center , istanbul , turkey . the diagnosis of ad was made according to the probable ad criteria proposed by the national institute of neurological and communicative disorders and stroke and the alzheimer s disease and related disorders association ( nincds - adrda),13 as well as the diagnostic and statistical manual of mental disorders , fourth edition.14 the control group participants were defined as having no demonstrable cognitively based limitations of activities of daily living , including employment , when applicable . rigorous criteria were used to exclude anyone with any type of symptomatic or severe brain - related neurologic or psychiatric illness . this was done by prospective interview of the participant and careful scrutiny of the medical records . examples of excluded conditions were mental retardation , epilepsy , cerebral infarction or hemorrhage , multiple sclerosis , brain tumors , major depressive disorders ( unipolar or bipolar ) , schizophrenia , traumatic brain injuries , and substance abuse . patients with more than mild mitral or aortic regurgitation and aortic or mitral stenosis were also excluded from the study . the nondemented ( nd ) participants were defined as having a mini - mental state examination13,14 score of 28 or higher , a clinical dementia rating of 0 , and functional assessment staging score of 2 or lower . all echocardiographic evaluations were performed with a transducer of 2.53.5 mhz and a ge vivid 3 echocardiography device ( general electric company , milwaukee , wi , usa ) . each patient was evaluated with m - mode , two - dimensional doppler , and tissue doppler echocardio graphy . the end - diastolic and end - systolic diameters of the left ventricle ( lv ) as well as the end - systolic diameter of the left atrium ( la ) were measured with m - mode echocardiography on the parasternal long axis view , in accordance with the american society of echocardiography guidelines.15 la volume was calculated with the ( a1a2a30.524 ) formula using the left atrial diameters measured from the parasternal long axis ( anteroposterior - a1 ) and apical four - chamber ( mediolateral - a2 , apicobasal - a3 ) views . mitral inflow velocities were evaluated by pulsed wave ( pw ) doppler echocardiography with the sample volume placed at the tip of the mitral leaflets from the apical four - chamber view . diastolic fillings were classified initially on the basis of the peak mitral flow velocity of the early rapid filling wave ( e ) , peak velocity of the late filling wave caused by atrial contraction ( a ) , e / a ratio , and deceleration time ( dt ) , which is the time interval for the peak e velocity to reach zero baseline . mitral inflow velocities were evaluated by pulsed wave ( pw ) doppler echocardiography with the sample volume placed at the tip of the mitral leaflets from the apical four - chamber view . the longitudinal motion of the mitral annulus has been shown to correlate with the rate of myocardial relaxation.15 the velocity of the mitral annulus can be recorded by the lv - pulsed tissue doppler imaging , which is an essential part of evaluation of diastolic function . tissue doppler imaging was performed in the apical four - chamber view using a 5 mm pulsed doppler sample volume with as minimal optimal gain as possible to obtain the best signal - to - noise ratio . the signal filter of the spectral pulsed doppler was adjusted until the nyquist limit was 1520 cm / s using a transducer of 3.54.0 mhz in frequency and we used minimal optimal gain to perform the tissue doppler echocardiography . the monitor sweep speed was set at 50100 mm / s to optimize the spectral display of myocardial velocities . on the apical four - chamber view , the pw doppler cursor was placed on the lateral mitral annulus of the left ventricle , septal mitral annulus , and right ventricle s tricuspid annulus , respectively , to obtain tissue doppler spectral images . the myocardial peak early ( ea ) and late diastolic ( aa ) velocities were obtained from these areas . the time passing from the beginning of the p wave on ecg to the aa wave on tissue doppler trace was accepted as the atrial conduction time . the atrial conduction times measured from mitral lateral ( mt ) , mitral septal ( ms ) , and tricuspid lateral ( tl ) annuli were named as ml - emd ( electromechanical delay ) , ms - emd , and tl - emd , respectively . the time difference between ml - emd and tl - emd we measured aortic diameters at a level 3 cm above the aortic cusps in long axis from the parasternal view . m - mode diameter measurements were made in systole ( point of maximal anterior motion of aorta ) and at end - diastole ( q wave on ecg ) . the means of three diameter measurements in sequential cardiac cycles were used for data analysis . systemic arterial blood pressure ( bp ) was measured at the right brachial artery by manual sphygmomanometer with the patient supine using an adequately sized cuff . pulse pressure ( pp ) was calculated by subtracting the diastolic bp from the systolic bp . the elastic properties of the aorta were indexed by calculation of aortic distensibility ( d ) , stiffness index ( si ) , and pressure - strain elastic modulus ( ep ) , and were as follows : ( 1)d=2(asad)/(ad[pspd ] ) , ( 2)si = ln(ps / pd)/([asad]/ad),and ( 3)ep=(pspd)/([asad]/ad),where as is aortic diameter at end - systole , ad is aortic diameter at end - diastole , ps is systolic bp , pd is diastolic bp , and ln is natural logarithm.1618 the continuous variables within group data were calculated as average standard deviation , and categorical variables as numbers and percentages . the student s t - test was used for comparison of parametric variables and the categorical variables were tested by pearson s test and fisher s exact test . binary logistic regression analysis was used to find the possible independent association between ad and study parameters . in multivariate regression analysis , p - values are two sided , and values < 0.05 were considered statistically significant . all statistical studies were carried out using statistical package for social sciences ( spss ) software ( v 16.0 for windows ; spss inc . , chicago , il , usa ) . all echocardiographic evaluations were performed with a transducer of 2.53.5 mhz and a ge vivid 3 echocardiography device ( general electric company , milwaukee , wi , usa ) . each patient was evaluated with m - mode , two - dimensional doppler , and tissue doppler echocardio graphy . the end - diastolic and end - systolic diameters of the left ventricle ( lv ) as well as the end - systolic diameter of the left atrium ( la ) were measured with m - mode echocardiography on the parasternal long axis view , in accordance with the american society of echocardiography guidelines.15 la volume was calculated with the ( a1a2a30.524 ) formula using the left atrial diameters measured from the parasternal long axis ( anteroposterior - a1 ) and apical four - chamber ( mediolateral - a2 , apicobasal - a3 ) views . mitral inflow velocities were evaluated by pulsed wave ( pw ) doppler echocardiography with the sample volume placed at the tip of the mitral leaflets from the apical four - chamber view . diastolic fillings were classified initially on the basis of the peak mitral flow velocity of the early rapid filling wave ( e ) , peak velocity of the late filling wave caused by atrial contraction ( a ) , e / a ratio , and deceleration time ( dt ) , which is the time interval for the peak e velocity to reach zero baseline . mitral inflow velocities were evaluated by pulsed wave ( pw ) doppler echocardiography with the sample volume placed at the tip of the mitral leaflets from the apical four - chamber view . the longitudinal motion of the mitral annulus has been shown to correlate with the rate of myocardial relaxation.15 the velocity of the mitral annulus can be recorded by the lv - pulsed tissue doppler imaging , which is an essential part of evaluation of diastolic function . tissue doppler imaging was performed in the apical four - chamber view using a 5 mm pulsed doppler sample volume with as minimal optimal gain as possible to obtain the best signal - to - noise ratio . the signal filter of the spectral pulsed doppler was adjusted until the nyquist limit was 1520 cm / s using a transducer of 3.54.0 mhz in frequency and we used minimal optimal gain to perform the tissue doppler echocardiography . the monitor sweep speed was set at 50100 mm / s to optimize the spectral display of myocardial velocities . on the apical four - chamber view , the pw doppler cursor was placed on the lateral mitral annulus of the left ventricle , septal mitral annulus , and right ventricle s tricuspid annulus , respectively , to obtain tissue doppler spectral images . the myocardial peak early ( ea ) and late diastolic ( aa ) velocities were obtained from these areas . the time passing from the beginning of the p wave on ecg to the aa wave on tissue doppler trace was accepted as the atrial conduction time . the atrial conduction times measured from mitral lateral ( mt ) , mitral septal ( ms ) , and tricuspid lateral ( tl ) annuli were named as ml - emd ( electromechanical delay ) , ms - emd , and tl - emd , respectively . the time difference between ml - emd and tl - emd was defined as the interatrial emd . we measured aortic diameters at a level 3 cm above the aortic cusps in long axis from the parasternal view . m - mode diameter measurements were made in systole ( point of maximal anterior motion of aorta ) and at end - diastole ( q wave on ecg ) . the means of three diameter measurements in sequential cardiac cycles were used for data analysis . systemic arterial blood pressure ( bp ) was measured at the right brachial artery by manual sphygmomanometer with the patient supine using an adequately sized cuff . pulse pressure ( pp ) was calculated by subtracting the diastolic bp from the systolic bp . the elastic properties of the aorta were indexed by calculation of aortic distensibility ( d ) , stiffness index ( si ) , and pressure - strain elastic modulus ( ep ) , and were as follows : ( 1)d=2(asad)/(ad[pspd ] ) , ( 2)si = ln(ps / pd)/([asad]/ad),and ( 3)ep=(pspd)/([asad]/ad),where as is aortic diameter at end - systole , ad is aortic diameter at end - diastole , ps is systolic bp , pd is diastolic bp , and ln is natural logarithm.1618 the continuous variables within group data were calculated as average standard deviation , and categorical variables as numbers and percentages . the student s t - test was used for comparison of parametric variables and the categorical variables were tested by pearson s test and fisher s exact test . binary logistic regression analysis was used to find the possible independent association between ad and study parameters . in multivariate regression analysis , p - values are two sided , and values < 0.05 were considered statistically significant . all statistical studies were carried out using statistical package for social sciences ( spss ) software ( v 16.0 for windows ; spss inc . , a total of 53 individuals were enrolled : 29 in the ad group ( 12 male and 17 female ; mean age standard deviation , 76.85.2 years ) and 24 in the nd control group ( 14 female and ten male ; mean age 77.16.7 years ) . the age difference between the ad and nd groups was not statistically significant ( p=0.65 ) . there were no significant differences between alzheimer patients and controls regarding age , sex , body mass index , and risk factors , including hypertension , diabetes mellitus , and smoking status . lv diastolic and systolic diameters , lv ejection fraction , and lv mass were comparable between the two groups . la anteroposterior diameters and la volumes were significantly higher in the ad group ( p=0.01 , p=0.01 , respectively ) . mitral valve pw doppler parameters were significantly different between the two groups , with lower e velocities and higher a velocities , dt , and ivrt in alzheimer patients ( p=0.01 for each comparison ) . mitral annular ea velocity was significantly lower in alzheimer patients , whereas aa velocity and e / ea ratio were similar between the groups . aortic diameters , parameters of aortic stiffness , and atrial conduction times are reported in table 3 . systolic aortic diameter was comparable between the groups ; however , diastolic aortic diameter was significantly higher in the alzheimer patients . ( 11.85.7 versus 3.31.1 ; p=0.01 ) and ep ( 139.538.7 versus 46.312.1 kpa ; p=0.01 ) were significantly higher , and d was significantly lower ( 1.510.75 versus 4.551.12 10 cm / dyn ; p=0.01 ) in the ad group . the atrial conduction times measured from the mitral valve s lateral ( ml ) and septal ( ms ) annuli were significantly higher in the alzheimer patients . however , the conduction time measured from the tl annulus was similar between the ad and nd groups . the interatrial emd was significantly higher in the ad group ( 16.111.1 versus 10.56.4 ms ; p=0.03 ) . in univariate binary logistic regression analysis , pp ( odds ratio [ or ] : 1.18 , 95% confidence interval [ ci ] : 1.071.306 ; p=0.01 ) , la volumes ( or : 1.20 , 95% ci : 1.081.35 ; p=0.01 ) , interatrial emd ( or : 1.07 , 95% ci : 1.011.146 ; p=0.04 ) , si ( or : 4.46 , 95% ci : 1.7111.64 ; p=0.01 ) , d ( or : 0.08 , 95% ci : 0.020.32 ; p=0.01 ) , and ep ( or : 1.12 , 95% ci : 1.041.21 ; p=0.01 ) were correlated with presence of ad . in multivariate regression analysis , using a model adjusted for age , sex , presence of hypertension , la volumes , and interatrial emd , d ( or : 0.09 , 95% ci : 0.020.41 ; p=0.01 ) and ep ( or : 1.12 , 95% ci : 1.021.23 ; p=0.02 ) were found to be independent correlates of ad . however , age , sex , presence of hypertension , la volume , interatrial emd , and si did not remain as independent correlates of the disease . the main findings of our study were that patients with ad have abnormal aortic stiffening compared to age- and sex - matched controls . even though lv diastolic parameters and atrial conduction times in ad patients were significantly different from control subjects , these parameters were not independent correlates of ad . in this study , we observed that patients with ad have altered diastolic function when compared to a control group . the intergroup differences of the e and a wave velocity , e / a ratio , dt , ivrt , la volume , and atrial conduction times measured from ml and ms annuli were statistically significant . mitral valve pw parameters were significantly different between the two groups , with lower e velocities and higher a velocities , dt , and ivrt in alzheimer patients . mitral annular ea velocity was significantly lower in alzheimer patients , whereas aa velocity and e / ea ratio were similar between the groups . our results from both the ad and control groups were consistent with borderline low e and borderline high a components of the diastolic filling velocities , resulting in an e / a ratio of less than 1.0 . this level of diastolic relaxation impairment is consistent with the age range of our study participants . however , our findings were associated with statistically significant impairment of diastolic parameters in the ad group when compared with the nd group . similar to our findings , belohlavek et al19 found that patients with ad have altered mitral inflow parameters and vortex formation time . however , they did not evaluate for the more sensitive tissue doppler parameters for diastolic functions that we observed in our study . in previous studies , it was shown that ad was associated with vascular risk factors and cardiovascular disease process.5,2023 hypertension , diabetes , stroke , and atherosclerosis have been shown to increase ad risk.7,24,25 during the aging process , heart , arteries , and brain suffer the continuous cumulative consequences of hemodynamic and mechanical stresses;19,2628 further , the myocardium undergoes structural changes including interstitial fibrosis and cellular disarray , which affects the diastolic functions negatively as a result of aging.2931 in this study , we observed that intraventricular blood transport rheologic processes are suboptimal and altered in patients with ad . although no causal relationship can be deduced from our data , it may be further speculated that the suboptimal hemodynamic efficiency could be the pathophysiologic link between the more pronounced lv relaxation dysfunction and ultimate brain cognitive dysfunction in patients with ad . however , these are preliminary observations and need to be taken cautiously because of the small number of patients involved in this study and the limitations in the accuracy of the clinical diagnosis of ad . in addition to conventional diastolic parameters we observed that patients with ad had increased atrial conduction times . yavuz et al32 found that atrial conduction times increase in hypertensive patients with diastolic dysfunction . also in yavuz et al s study,32 the atrial conduction times measured from ml and ms and also interatrial emd were longer in the ad group , which can be associated with more impaired diastolic function . in our study , we observed that pp , ep , d , and si were correlated with presence of ad . the measurements in our study were made by conventional echocardiography , and this is the key difference of our study from others.10,11,28,33 similar to our findings , hanon et al10 observed that pw velocity ( pwv ) was significantly higher in subjects with vascular dementia or ad than in those without cognitive impairment , and they pointed out that arterial stiffness appeared to be an independent determinant not only of vascular dementia , but also of ad . a previous study by bateman et al28 showed that early ad is characterized by reduced blood flow and reduced compliance . in contrast to our observations , a previous study by dhoat et al11 which compared vascular compliance and arterial stiffness between vascular dementia , ad , and control groups by measuring central arterial compliance , augmentation index , and pwv , concluded that arterial stiffness in patients with ad is no different from that in controls . several mechanisms may explain our findings of an association between increased aortic stiffness and ad . aortic stiffness can cause an increase in central pp , which may influence arterial remodeling at the site of the extracranial and intracranial arteries . indeed , pp and arterial stiffness have been related to atherosclerosis or arteriosclerosis in large34,35 and small36 vessels . an increased pp has been associated with the prevalence and severity of cerebral white matter lesions,37 and , in a recent study , aortic stiffness appeared as an independent predictor of stroke in patients with essential hypertension.38 our study indicates an independent correlation between aortic stiffness and ad , supporting previous studies suggesting that functional changes of the arterial system10,11,28,33 could play a role in the pathogenesis of alzheimer dementia . this study was a cross - sectional study without long - term follow - up , and the prognostic importance of these echocardiographic parameters is not known . in this study , we observed that patients with ad have altered diastolic function when compared to a control group . the intergroup differences of the e and a wave velocity , e / a ratio , dt , ivrt , la volume , and atrial conduction times measured from ml and ms annuli were statistically significant . mitral valve pw parameters were significantly different between the two groups , with lower e velocities and higher a velocities , dt , and ivrt in alzheimer patients . mitral annular ea velocity was significantly lower in alzheimer patients , whereas aa velocity and e / ea ratio were similar between the groups . our results from both the ad and control groups were consistent with borderline low e and borderline high a components of the diastolic filling velocities , resulting in an e / a ratio of less than 1.0 . this level of diastolic relaxation impairment is consistent with the age range of our study participants . however , our findings were associated with statistically significant impairment of diastolic parameters in the ad group when compared with the nd group . similar to our findings , belohlavek et al19 found that patients with ad have altered mitral inflow parameters and vortex formation time . however , they did not evaluate for the more sensitive tissue doppler parameters for diastolic functions that we observed in our study . in previous studies , it was shown that ad was associated with vascular risk factors and cardiovascular disease process.5,2023 hypertension , diabetes , stroke , and atherosclerosis have been shown to increase ad risk.7,24,25 during the aging process , heart , arteries , and brain suffer the continuous cumulative consequences of hemodynamic and mechanical stresses;19,2628 further , the myocardium undergoes structural changes including interstitial fibrosis and cellular disarray , which affects the diastolic functions negatively as a result of aging.2931 in this study , we observed that intraventricular blood transport rheologic processes are suboptimal and altered in patients with ad . although no causal relationship can be deduced from our data , it may be further speculated that the suboptimal hemodynamic efficiency could be the pathophysiologic link between the more pronounced lv relaxation dysfunction and ultimate brain cognitive dysfunction in patients with ad . however , these are preliminary observations and need to be taken cautiously because of the small number of patients involved in this study and the limitations in the accuracy of the clinical diagnosis of ad . in addition to conventional diastolic parameters we observed that patients with ad had increased atrial conduction times . yavuz et al32 found that atrial conduction times increase in hypertensive patients with diastolic dysfunction . also in yavuz et al s study,32 the atrial conduction times measured from ml and ms and also interatrial emd were longer in the ad group , which can be associated with more impaired diastolic function . in our study , we observed that pp , ep , d , and si were correlated with presence of ad . the measurements in our study were made by conventional echocardiography , and this is the key difference of our study from others.10,11,28,33 similar to our findings , hanon et al10 observed that pw velocity ( pwv ) was significantly higher in subjects with vascular dementia or ad than in those without cognitive impairment , and they pointed out that arterial stiffness appeared to be an independent determinant not only of vascular dementia , but also of ad . a previous study by bateman et al28 showed that early ad is characterized by reduced blood flow and reduced compliance . in contrast to our observations , a previous study by dhoat et al11 which compared vascular compliance and arterial stiffness between vascular dementia , ad , and control groups by measuring central arterial compliance , augmentation index , and pwv , concluded that arterial stiffness in patients with ad is no different from that in controls . several mechanisms may explain our findings of an association between increased aortic stiffness and ad . aortic stiffness can cause an increase in central pp , which may influence arterial remodeling at the site of the extracranial and intracranial arteries . indeed , pp and arterial stiffness have been related to atherosclerosis or arteriosclerosis in large34,35 and small36 vessels . an increased pp has been associated with the prevalence and severity of cerebral white matter lesions,37 and , in a recent study , aortic stiffness appeared as an independent predictor of stroke in patients with essential hypertension.38 our study indicates an independent correlation between aortic stiffness and ad , supporting previous studies suggesting that functional changes of the arterial system10,11,28,33 could play a role in the pathogenesis of alzheimer dementia . this study was a cross - sectional study without long - term follow - up , and the prognostic importance of these echocardiographic parameters is not known . our findings suggest that patients with ad , the most common form of dementia , are more likely to have diastolic dysfunction and longer atrial conduction times . more importantly , our study has shown that d and ep , which are the basic parameters of aortic stiffness , were independently correlated with presence of ad . although we could not detect a causal relationship in this study , subclinical cardiac involvement in ad warrants further investigation . further prospective studies are needed to clarify the role of these findings during disease development , progress , and management .

thrombocytosis is often an incidental finding seen in 3550% of cases and the cause determination creates a diagnostic challenge . extreme thrombocytosis is rare and seen in 25.8% patients only . among the various causes of increased platelet count , surgical procedures have attracted much attention in both experimental and clinical domain . the appearance of thrombocytosis after surgery needs to be diagnosed to establish the type of thrombocytosis ( clonal or reactive ) , as treatment and prognosis are quite different between them . this case report is vital because of two reasons : first , the increase in platelet count is difficult to rationalize than many of the other thrombocytoses , such as those related to primary augmentation of the function of the bone marrow ; second , the association of platelets with the clotting process has led to the belief that their increase after a surgical procedure is connected with the occurrence of postoperative thrombosis . this case presents an interesting finding from a patient who has undergone major abdominal surgery and has shown an unexpected perpetual increase in platelet count . backgroundalzheimer s disease ( ad ) is closely linked to cardiovascular risk factors.methodsechocardiographic studies were performed , including left ventricular diastolic functions , left and right atrial conduction times , and arterial stiffness parameters , namely stiffness index , pressure - strain elastic modulus , and distensibility , on 29 patients with ad and 24 age - matched individuals with normal cognitive function.resultsthe peak mitral flow velocity of the early rapid filling wave ( e ) was lower , and the peak velocity of the late filling wave caused by atrial contraction ( a ) , deceleration time of peak e velocity , and isovolumetric relaxation time were higher in the ad group . the early myocardial peak ( ea ) velocity was significantly lower in ad patients , whereas the late diastolic ( aa ) velocity and e / ea ratio were similar between the two groups . in alzheimer patients , stiffness index and pressure - strain elastic modulus were higher , and distensibility was significantly lower in the ad group compared to the control . interatrial electromechanical delay was significantly longer in the ad group.conclusionour findings suggest that patients with ad are more likely to have diastolic dysfunction , higher atrial conduction times , and increased arterial stiffness compared to the controls of same sex and similar age .

the subplate forms a transient layer in the developing cerebral cortex and consists of migratory and postmigratory neurons , dendrites , axons , growth cones , synapses and glial cells . the subplate is located between the intermediate zone and the cortical plate , which during further development differentiates into the neocortical layers ii to vi ( figure 1a ) . kostovic and molliver were the first who identified the subplate as a distinct layer in the embryonic human cerebral cortex ( kostovic and molliver , 1974 ) . a subplate can be identified in all mammals although its relative thickness , developmental profile and persistence in adulthood vary among species . anatomical data indicate an evolutionary difference in the ontogenetic fate of the subplate . in the rat and other rodents many subplate cells survive into adulthood forming layer vib or vii ( aboitiz and montiel , 2007 ) . prominent species differences also exist in the relative thickness of the subplate , which increased during evolution . in humans the subplate develops to approximately six times the thickness of the cortical plate around 29 weeks of gestation ( mrzljak et al . , 1990 ) , whereas in rodents it remains a relatively thin layer during development ( uylings et al . , 1990 ) . the majority of the subplate cells are born early before the first cortical plate neurons ( luskin and shatz , 1985 ; valverde et al . , 1989 ) . in rodents subplate neurons may be also generated later in development ( hevner et al . , 2004 ) . a substantial proportion of subplate cells are not born in the ventricular neuroepithelium , but instead originate in the medial ganglionic eminence and follow a tangential migratory route to their positions in the developing cortex ( lavdas et al . , 1999 ) . subplate cells represent a rather heterogeneous neuronal population according to their morphology , neurotransmitter identity and connectivity ( for review allendoerfer and shatz , 1994 ) . ( a ) prenatal development of the human cerebral cortex as suggested by bystron et al . ( 2008 ) . drawing from pasko rakic . photograph shows coronal brain section stained with cresyl violet of a gestational week 18 human neocortex . approximate embryonic day ( e ) and gestational week ( gw ) are given for each developmental stage . cp , cortical plate ; iz , intermediate zone ; mz , marginal zone ; pp , preplate ; sp , subplate ; svz , subventricular zone ; ( sg ) , subpial granular layer ( part of mz ) ; vz , ventricular zone . ( b ) morphological properties of biocytin - stained subplate neurons in newborn rat cerebral cortex . ( b1 ) postnatal day ( p ) three horizontal bitufted cell with large fusiform soma and primary dendrites oriented parallel to the pial surface . ( b3 ) p3 multipolar cell with extensive dendritic arborization within sp and layers v / vi . ( b4 ) p2 inverted pyramidal neuron with triangular soma and apical dendrite oriented towards white matter . scale bar in ( b4 ) corresponds to ( b1b4 ) and pial surface is located toward the top in all photomicrographs of ( b ) . reproduced and modified with permission from ( bystron et al . , 2008 ) ( a ) , ( kostovic et al . , 2002 ) [ photograph in ( a ) ] and from ( hanganu et al . , 2002 ) ( b ) . subplate cells play important roles in the structural and functional organization of the cerebral cortex and in early necortical plasticity . axons arising from subplate neurons pioneer the corticofugal pathway and have been proposed to form a cellular scaffold for guiding thalamocortical axons ( mcconnell et al . 1990 ) ( for review allendoerfer and shatz , 1994 ; molnr , 1998 ) . thalamic axons ( lund and mustari , 1977 ; rakic , 1977 , 1983 ) and early deletion of subplate neurons in kitten visual cortex prevents the segregation of thalamocortical axons within layer iv and the formation of ocular dominance columns ( ghosh and shatz , 1992 ; kanold et al . , 2003 ) . furthermore , subplate cells regulate the maturation of gabaergic synaptic transmission and establish the balance between excitation and inhibition in the developing neocortical network ( kanold and shatz , 2006 ) . subplate neurons reveal a large variety of morphologies ( figure 1b ) ( hanganu et al . , 2002 ) . inverted pyramidal - like and horizontal cells as well as polymorphic neurons with different shapes and spiny or smooth dendrites have been classified as subplate neurons ( kostovic and rakic , 1980 ; wahle et al . , 1987 ; valverde et al . , 1989 ; kostovic and rakic , 1990 ) due to their earlier generation and more mature developmental stage , subplate neurons show a relatively extensive dendritic tree when compared to the more immature pyramidal neurons of the cortical plate ( mrzljak et al . , 1992 ) . descending dendrites from subplate neurons may invade the underlying intermediate zone and ascending dendrites may extend into the cortical plate ( del ro et al . , 2000 ) . subplate neurons reveal markers for gaba or glutamate and may co - express various peptides ( chun et al . , 1987 ; the morphological and neurochemical heterogeneity also explains , why various attempts failed to identify a specific marker for subplate neurons ( wahle et al . , 1994 ) . ( 2009 ) succeeded in identifying a number of novel markers for murine subplate cells , which may soon allow the definition of different subpopulations of subplate neurons . subplate cells participate in local and long - distance axonal connections indicating that these neurons may function as local circuit as well as projection neurons . they show a dense axonal arborization within the subplate , but also project to the marginal zone / layer i ( clancy and cauller , 1999 ) and to the cortical plate , where they form axonal collaterals within layer iv ( friauf et al . , the majority of the subplate neurons projecting into the cortical plate reside in the upper half of the subplate and provide a glutamatergic synaptic input to the developing cortical plate , including the layer iv neurons ( friauf et al . , 1990 ; long - distance axons from subplate neurons invade the thalamus during early stages of corticogenesis and form an axonal scaffold for the establishment of cortical efferent and afferent projections ( mcconnell et al . , 1989 , 1994 ; kim et al . , 1991 ; de carlos and o'leary , 1992 ) . beside these local and long - distance projections arising from glutamatergic subplate neurons , gabaergic subplate cells also project to both neighbouring and more distant neocortical regions and form a corticocortical synaptic network ( tomioka et al . , 2005 ; higo et al . , 2007 ) . since gaba may act as an excitatory neurotransmitter during early cortical development ( for review ben - ari et al . , 2007 ) , the postsynaptic action of gabaergic subplate neurons may be also depolarizing ( figure 6 in hanganu et al . , 2002 ) . ultrastructural studies of subplate cells in various species have demonstrated symmetrical as well as asymmetrical synapses with relatively mature properties ( kostovic and rakic , 1980 , 1990 ; chun and shatz , 1988 ; herrmann et al . , 1994 ) , indicating that subplate neurons receive gabaergic as well as glutamatergic synaptic inputs . as suggested by kostovic and rakic ( 1980 ) , glutamatergic inputs onto subplate neurons may arise from the thalamus and other neocortical areas , whereas gabaergic synaptic inputs may originate from gabaergic interneurons in the subplate . thalamocortical synaptic contacts with spines and shafts of subplate neuron dendrites have been demonstrated in the neonatal ferret ( herrmann et al . , 1994 ) and a dense network of corticocortical fibers have been reported in the subplate of the embryonic mouse ( crandall and caviness , 1984 ) . n - methyl - d - aspartate ( nmda ) , -amino-3-hydroxy-5-methylisoxazole-4-propionic acid ( ampa ) and kainate receptors and the essential subunits for their receptor function have been demonstrated in the subplate of various species , suggesting the presence of functional glutamatergic synapses in subplate neurons ( herrmann , 1996 ; aoki , 1997 ; catalano et al . , gabaa receptors ( huntley et al . , 1990 ) and gabaa receptor subunits ( meinecke and rakic , 1992 ) in the subplate indicate that functional gabaergic synaptic inputs should also be present in subplate neurons . these morphological , ultrastructural and immunohistochemical data are complemented by functional studies on the properties of the subplate and single subplate cells in different mammalian species . in contrast to the heterogeneity in morphological and chemical appearance , electrophysiological recordings from single subplate neurons in rodents demonstrate rather homogeneous functional properties . whole - cell patch - clamp recordings from subplate cells in newborn rodent neocortical slices revealed resting membrane potentials and input resistances in the range of 55 mv and 11.2 g , respectively ( luhmann et al . , hirsch and luhmann , 2008 ) . in response to sustained depolarization by intracellular current injection , subplate neurons are capable of firing overshooting and repetitive action potentials at frequencies exceeding 40 hz ( figures 2c and 3a ) . similar results were obtained from subplate neurons in acute neocortical slices harvested postmortem from human fetal brain at gestational week 1622 ( moore et al . , 2009 ) . ( a ) intracellular biocytin staining of one subplate neuron ( yellow arrow ) in the p3 mouse neocortex results in columnar labeling of several dye - coupled cells in the subplate and in the cortical plate.(b ) photograph of two simultaneously recorded p1 subplate neurons , which were both stained with biocytin . ( c ) paired current - clamp recordings of the cells shown in ( b ) . black traces were obtained from the cell marked by black arrow in ( b ) and red traces are from the cell marked by red arrow . ( c1 ) injection of a hyperpolarizing or suprathreshold depolarizing current pulse into the cell marked by black arrow causes voltage deflections in the gap junction coupled neuron . ( c2 ) vice versa , injection of a hyperpolarizing or suprathreshold depolarizing current pulse into the cell marked by red arrow causes voltage deflections in the other neuron . cortical surface in ( a ) and ( b ) is up , action potentials in ( c ) are truncated . the hyperpolarizing responses in the lower traces are averages of four recordings . reproduced and modified with permission from ( dupont et al . , 2006 ) . ( a ) typical action potential firing pattern of a subplate neuron in the cerebral cortex of a p1 rat . ( b ) excitatory postsynaptic currents ( epscs ) recorded in a p3 subplate cell at a holding potential of 70 mv and elicited by selective electrical stimulation of the thalamocortical input ( see c ) . synaptic responses were obtained under control conditions in normal extracellular bathing solution and after application of 10 m cnqx to block ampa / kainate receptors . the inset shows the unimodal latency distribution of 50 thalamocortical epscs recorded from one spn . ( c ) photomicrograph of biocytin - stained thalamocortical projections in a 400-m thick coronal slice from a p1 rat . asterisk shows extracellular injection site of biocytin crystal and white circles mark position of bipolar electrode to selectively activate the thalamocortical input . ( d ) schematic diagram illustrating the distribution of postsynaptic receptors on a subplate neuron for monosynaptic inputs arising from the thalamus ( thal . ) , the cortical plate ( cp ) including layers v / vi and within the subplate ( sp ) . note that synaptic inputs from other subplate neurons can lead to activation of depolarizing gabaa receptors and nmda receptors activated at negative membrane potentials . reproduced and modified with permission from ( luhmann et al . , 2000 ) ( a ) ( hanganu et al . , 2002 ) ( b d ) . in comparison to other neurons in the immature cerebral cortex , subplate cells also reveal the most mature properties in action potential characteristics and in the biophysical properties of voltage - dependent sodium and calcium currents . these observations have been made in developing rodent ( luhmann et al . , 2000 ) as well as in human cerebral cortex ( moore et al . , 2009 ) , indicating that these relatively mature functional properties enable subplate cells to transmit afferent neuronal activity faithfully to the developing cortical plate . intracellular labeling of single subplate cells with fluorescent dyes or biocytin revealed an extensive neuronal network of dye - coupled neurons in the subplate and cortical plate ( figure 2a ) . in newborn rats , on average about nine neurons are dye - coupled to a single subplate cell and these gap junction coupled networks are often organized in a columnar manner ( dupont et al . , 2006 ) . whole - cell patch - clamp recordings from pairs of dye - coupled subplate neurons ( figure 2b ) allowed a more detailed electrophysiological characterization of the electrical synapses connecting subplate neurons ( figure 2c ) . the average coupling conductance amounted to about 1.2 ns ( dupont et al . , 2006 ) . these data indicate that subplate cells are strongly coupled via electrical synapses and form a functional columnar syncytium with neurons located in the cortical plate . it is tempting to speculate that this early columnar organization results from the radial , column - like neuronal migration of newly generated neurons into the developing neocortex ( noctor et al . , 2004 ) , which is also controlled by gap junctional coupling ( elias et al . , 2007 ) electrophysiological and optical imaging recordings further support the hypothesis that subplate neurons are well integrated in the developing cerebral cortex . in vitro intracellular recordings and current - source density analyses in late embryonic and early postnatal kitten visual cortex demonstrated that subplate neurons receive functional excitatory synaptic inputs from axons that course in the developing white matter ( friauf et al . subplate cells in newborn rat somatosensory cortical slices reveal a substantial amount of spontaneous postsynaptic currents ( spscs ) with different kinetics and pharmacological profile demonstrating that subplate neurons receive functional synaptic inputs mediated by ampa , nmda and gabaa receptors ( hanganu et al . , 2001 ) . a more detailed analysis of the synaptic inputs onto subplate cells could be obtained by electrical stimulation of specific afferent axonal projections ( figures 3b , c ) ( staiger et al . , 1999 ) . brief electrical stimulation of the thalamocortical projection elicits in immature rat and cat subplate neurons a fast and reliable excitatory postsynaptic potential / current ( epsp / c ) , which can blocked by the ampa / kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione ( cnqx ) ( friauf et al . , 1990 ; hanganu et al . , elicits a cnqx - insensitive component , which can be blocked by the nmda receptor antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ( cpp ) , demonstrating that the thalamic input also activates functional nmda receptors on subplate cells ( hanganu et al . , 2002 ) . these electrophysiological observations from single subplate neurons are further supported by current source - density analyses performed in late embryonic and early postnatal kitten ( friauf and shatz , 1991 ) and rat cerebral cortex ( molnr et al . , 2003 ) , demonstrating that subplate cells in these species receive a functional thalamic input at earliest stages of neocortical development . recording of optical images with voltage - sensitive dyes in neocortical slices of prenatal rats have shown that there is a few days delay between the arrival of thalamocortical axons at the subplate at e16 and the appearance of functional thalamocortical synaptic transmission at e19 ( higashi et al . , 2002 ) . beside a glutamatergic thalamocortical input with relatively mature functional properties ( short delay , fast kinetics , reliable responses ) , subplate cells in newborn rodents receive additional intracortical synaptic inputs from various presynaptic sources ( figure 3d ) . local glutamatergic synaptic inputs arise from pyramidal neurons in the cortical plate and from glutamatergic subplate cells , which both activate postsynaptic ampa / kainate and nmda receptors ( hanganu et al . , this intra - subplate glutamatergic input differs from the thalamocortical input . whereas the intra - subplate input reveals a pronounced facilitation when repetitively activated at 1040 hz , the thalamocortical input is rather stable or suppressed at these stimulation frequencies ( figure 4 ) ( hirsch and luhmann , 2008 ) . the intra - subplate synaptic input can sustain high stimulation frequencies and may boost the thalamocortical input , thereby enhancing activity in the developing neocortical circuit ( hirsch and luhmann , 2008 ) . anatomical studies in rodent and human immature cerebral cortex indicate that glutamatergic synaptic inputs most likely also arise from other neocortical sources via corticocortical connections ( ivy and killackey , 1981 ; kostovic and jovanov - milosevic , 2006 ) , but the functional properties of these long - distance synaptic inputs onto subplate cells are currently unknown . input - specific differences in temporal summation of nmda receptor - mediated epsps recorded in subplate neurons of the newborn mouse cerebral cortex . recordings were performed in the presence of cnqx and bicuculline methiodide to isolate nmda receptor - mediated epsps . ( a ) five successive stimuli to the thalamic ( left ) or subplate ( right ) input reveals prominent temporal summation and repetitive action potentials ( truncated ) of the subplate input at frequencies between 10 and 40 hz . ( b ) synaptic response to 20-hz subplate stimulation at higher resolution [ same trace as in ( a ) ] . ( c ) quotient between nmda receptor - epsp integral and control integral ( determined at 0.03 hz ) for 13 subplate neurons . reproduced with permission from hirsch and luhmann ( 2008 ) . subplate cells in rodents receive a gabaergic synaptic input from neighbouring gabaergic neurons located in the subplate and probably also in the cortical plate ( figure 3d ) ( hanganu et al . , 2002 ) . however , as in other immature brain structures , this gabaergic input most likely has a pure excitatory postsynaptic effect ( figure 6 in hanganu et al . an excitatory effect has been demonstrated in subplate cells also for glycine and taurine ( kilb et al . , 2008 ) . subplate neurons are not only well integrated in the developing cortical circuit , but during certain developmental periods they also receive a very selective input from neuromodulatory brain structures . both in primates ( for review ( rakic , 1995 ) as well as in rodents ( calarco and robertson , 1995 ; mechawar and descarries , 2001 ) the subplate is specifically innervated by cholinergic fibers arising from the basal forebrain and from monoaminergic inputs . the activation of postsynaptic nicotinic acetylcholine receptors elicits in subplate cells a marked depolarization which is largely mediated by the activation of alpha4/beta2 receptors ( hanganu and luhmann , 2004 ) . the responsiveness to activation of muscarinic acetylcholine receptors ( machr ) is more complex and reveals a remarkable oscillatory discharge mode of subplate cells ( hanganu et al . , 2009 ) . application of muscarine to neocortical slices from newborn rats induces in subplate cells repetitive burst discharges with burst frequencies in the range of 20 hz ( figure 5a ) ( hanganu et al . , 2009 ) . similar burst patterns can be recorded from subplate neurons when the tissue concentration of acetylcholine is raised by application of the cholinesterase inhibitor neostigmine ( figure 5b ) . these electrophysiological data are further supported by calcium imaging experiments , which demonstrate that activation of muscarinic receptors induces repetitive ca - transients which are highly coordinated within the subplate ( figure 1 in hanganu et al . , 2009 ) . subplate cells , which express the m1m5 subunits of the machr , are instantly and massively excited upon activation of postsynaptic muscarinic receptors and switch into an oscillatory burst firing mode ( figure 5 ) . since subplate cells are densely coupled via electrical ( figure 2 ) and chemical synapses ( figure 3d ) , these cellular oscillations are synchronized and amplified within the intra - subplate network . gap junctional coupling , depolarizing gaba actions and a tonic non - synaptic gaba release contribute to the generation and maintenance of the cholinergic network oscillations ( figure 9 in hanganu et al . , 2009 ) . subplate neurons in newborn rat cerebral cortex reveal repetitive burst discharges upon activation of postsynaptic cholinergic receptors . ( a ) current - clamp whole - cell recording from a p3 subplate cell at a holding potential of 70 mv . note the long - lasting burst discharge after a single 30-s long bath application of muscarine ( marked by a black bar ) . frequency power spectrum of the action potential discharge within one representative burst from the trace above . ( b ) current - clamp whole - cell recording from a p2 subplate neuron at a holding potential of 70 mv displaying the effects of 10 m neostigmine , which blocks endogenous acetylcholine esterase and thereby increases the concentration of acetylcholine in the neocortical slice . frequency power spectrum of the action potential discharge within burst from the upper trace in ( b ) . the crucial role of the subplate in generating cholinergic network oscillations is supported by experiments on thick ( 8001000 m ) neocortical slices from newborn rodents . only slices with an intact subplate reveal network oscillations upon activation of muscarinic receptors with carbachol ( dupont et al . , 2006 ) . when the subplate is removed , slices do not show any carbachol - induced network oscillations ( figure 6 ) . furthermore , selective electrical stimulation of the subplate in thick neocortical slices from newborn mice using a multi - electrode array ( mea ) also evokes large - scale network oscillations ( figure 7 ) ( sun and luhmann , 2007 ) . in a recent study on mouse neocortical slice cultures , moody and coworkers suggested that the subplate may act as a pacemaker region to generate propagating waves of spontaneous activity in the neonatal cerebral cortex ( lischalk et al . , 2009 ) . , 2006 ; sun and luhmann , 2007 ) or by the sensory input , i.e. the retina ( hanganu et al . , 2006 ) or the whiskers ( yang et al . , 2009 ) . at early developmental stages , the sensory input from the periphery firstly reaches the subplate and is subsequently transmitted to the cortex ( friauf and shatz , 1991 ) . during this developmental period , role of the subplate in triggering carbachol - induced network oscillations in the neonatal mouse cerebral cortex . ( a ) field potential response to carbachol application in a coronal 800-m thick cortical slab preparation with an intact subplate as shown in nissl stained section below . ( b ) lack of carbachol - induced activity in a cortical slab preparation , in which the subplate was eliminated . local electrical stimulation of the subplate elicits propagating network oscillations in the developing cerebral cortical network of the newborn mouse . ( a ) photograph of p3 mouse nissl - stained coronal slice with cortical layers and position of 60-channel multi - electrode array ( mea ) . ( b ) mea recordings from the neocortical slice shown in ( a ) with site of bipolar stimulation in the subplate ( 80 a , 200-s duration , 50 hz , 10 times ) . spacing between electrodes ( c ) recording at electrode number 84 [ dotted red rectangle in ( a ) ] shown at higher resolution with corresponding wavelet analysis and fourier spectrum . reproduced with permission from sun and luhmann ( 2007 ) . electrophysiological and calcium imaging recordings in acute neocortical slices from newborn rodents ( hanganu et al . , 2009 ) as well as calcium imaging data obtained in neocortical cell cultures ( voigt et al . , 2001 ) indicate that gabaergic subplate cells play a central role in generating this neuronal network activity . voigt et al . ( 2001 ) estimated that a minimal number of two gabaergic subplate neurons per square millimeter are required for the occurrence of synchronous network activity . the unique structural and functional properties enable subplate cells to receive , synchronize and amplify the afferent and intrinsic synaptic inputs . it is tempting to speculate on the downstream effects of these early network oscillations in cortical maturation ? it has been demonstrated that brain derived neurotrophic factor ( bdnf ) is released from synaptically localized secretory granules following burst stimulation at 2050 hz ( balkowiec and katz , 2000 ; hartmann et al . , 2001 ) ( for review lessmann et al . , 2003 ) . if subplate - driven oscillations contribute to the local secretion of bdnf , the released bdnf may strengthen the synaptic connectivity between neurons in these early ensembles . beside strengthening synaptic connections via local bdnf release , we recently demonstrated that the rate of apoptosis in organotypic slice cultures of the neonatal mouse cerebral cortex is regulated by electrical activity patterns , which resemble in many aspects the subplate - driven activity ( heck et al . , 2008 ) . this activity - dependent regulation of neuronal apoptosis was at least partly mediated by activation of the bdnf receptor trkb , indicating that subplate - driven network oscillations may also influence programmed cell death via activity - dependent bdnf release . a large proportion of newly generated gabaergic interneurons arising from the medial ganglionic eminence and pyramdial neurons from the ventricular zone must migrate through the subplate on their way to the developing cortical plate ( kriegstein and noctor , 2004 ) . gabaergic and glutamatergic subplate neurons partly release their neurotransmitter in a paracrine , non - synaptic manner ( hanganu et al . , 2009 ) , thereby regulating the neuronal migration pattern ( reiprich et al . , 2005 ; manent et al . , 2005 , 2006 ; . neurotransmitter release in the subplate may rise substantially during oscillatory network activity , thereby activating low affinity or extrasynaptic receptors causing alterations in neuronal migration ( denter et al . , 2009 ) . on the basis of the currently available data we suggest the following model ( figure 8) : during early cortical development , in most mammals before birth , subplate cells with relatively mature structural properties ( elaborated dendritic tree , complex axonal projections , mature symmetrical and asymmetrical synapses ) receive a functional glutamatergic synaptic input from specific thalamic nuclei and a selective input from neuromodulatory systems ( e.g. cholinergic inputs from the basal forebrain ) . in rodents , subplate neurons are densely interconnected via electrical and chemical synapses and upon activation of muscarinic ( and probably also other metabotropic receptors ( wagner and luhmann , 2006 ) discharge in repetitive 20 hz bursts ( lower inset in figure 8) . gabaergic subplate neurons releasing gaba in a synaptic ( 1 in figure 8) and tonic non - synaptic manner ( 2 in figure 8) facilitate the generation of oscillatory network activity ( hanganu et al . , 2009 ) . intra - subplate connections arising from glutamatergic subplate cells may boost the subplate activity in the 1040 hz frequency range ( hirsch and luhmann , 2008 ) . subplate cells are dye - coupled to cortical plate neurons in a columnar manner ( dupont et al . , 2006 ) and faithfully transmit synchronized 1020 hz network oscillations generated and amplified in the subplate to the cortical plate and marginal zone ( upper inset in figure 8) ( dupont et al . , 2006 ; wagner and luhmann , 2006 ; the subplate receives a transient and selective synaptic input from neuromodulatory systems , from the sensory periphery via specific thalamic nuclei and via corticocortical fibers from other neocortical areas . subplate cells are capable to fire repetitive action potentials ( figure 3a ) and upon activation of cholinergic postsynaptic receptors discharge in repetitive bursts ( lower inset and figure 5 ) . gabaergic subplate neurons release gaba in an activity - dependent ( 1 ) and tonic ( 2 ) manner thereby inducing a postsynaptic excitatory response . subplate neurons are not only coupled via these excitatory synaptic circuits , but also via gap junctions ( figure 2 ) . chemical and electrical synapses contribute to the generation of subplate - driven oscillatory activity which is transmitted to the gap junctional coupled network in the cortical plate ( figure 2a ) and marginal zone and induces 1020 hz neocortical network oscillations ( upper inset ) . elimination or interruption of the subplate prevents the generation of the subplate - driven synchronized activity patterns and disturbs the maturation of the columnar architecture ( ghosh and shatz , 1992 ; kanold et al . , 2003 ; kanold and shatz , 2006 ) . under normal conditions and with further development , subplate cells disappear by apoptosis ( ferrer et al . , 1990 ; price et al . , 1997 ; arias et al . , 2002 ) or transform into white matter interstitial cells ( valverde and facal - valverde , 1988 ; valverde et al . , 1995 ) or layer vib ( layer vii ) neurons ( woo et al . , 1991 ; reep , 2000 ) in the normal mature cerebral cortex , subplate neurons have lost their capabilities to receive and amplify incoming neuronal activity and to generate synchronized network oscillations . however , as already suggested by jones ( 1995 ) , disturbances in the pattern of programmed cell death in the subplate may cause a failure to establish normal patterns of connections in the overlying cerebral cortex , leading to long - term neurological deficits such as schizophrenia . abnormal placement of ( surviving subplate ? ) neurons in the white matter and atypical circuits have been observed in the prefrontal cortex of schizophrenic patients ( akbarian et al . , 1996 ; it has been further suggested that subplate - like neurons may persist in cortical dysplasia and contribute to the manifestation of pharmacoresistant epilepsy in adults ( cepeda et al . , 2007 ) . interestingly in the resectioned human tissue gaba application induced depolarizing postsynaptic responses and spontaneous gabaergic synaptic potentials even elicited action potentials ( cepeda et al . , 2007 ) . in summary , experimental data indicate that subplate cells , most likely gabaergic subplate neurons , play an important role in the generation of early synchronized network activity and in the normal development of the neocortical network and columnar architecture . clinical evidence suggests that subplate cells in the mature cortex contribute to the manifestation of abnormal neuronal circuits , pathological activities and long - term neurological deficits . the authors declare that the research presented in this paper was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . reflection is growing in importance as a means to promote a learner - centered environment where the learner is encouraged to learn through the practice of nursing with subsequent reflection . when students are in uncertain new situations , they are expected to use focused thought to apply learned principles to clinical situations and critically evaluate their performance and decisions , rather than focusing on technical knowledge in isolation . the national league for nursing has reiterated the importance of both reflective and critical thinking through their designation as core competencies for nurse educators . clinical journals are one of the strategies used throughout the undergraduate nursing curricula to facilitate and guide students ' reflective thinking processes . reflective journals are considered by ( most ) faculty members to be essential in fostering an understanding of course concepts and application of concepts to clinical practice ; however , students continually question their worth in their anecdotal comments and in formal student course evaluations . is student dissatisfaction based on reflective journaling in general , the format within an individual course or modality , or some other characteristic of the individual student ? it is possible that reflective journals are not consistently reaching the anticipated critical reflection / thinking outcomes that are desired for all students . the purposes of the study were to determine ( a ) the critical attributes of a reflective journal and relative levels of each attribute , ( b ) the relative importance of each attribute as perceived by the students , and ( c ) the perception of relative importance for student subgroups . the reflective learning process for one entering a profession , particularly one entering the health care field , is a very personal experience . the day - to - day experiences in the clinical setting often challenge strongly held beliefs . educators rightfully focus on developing learning tools that include reflection on learned course content , on clinical experiences , on personal values and on their role as the novice nurse . at the foundation of the journaling process reflection , via journal writing , has been an integral component of clinical learning for many years . most educators agree that reflective journaling is valuable and contributes to learning ; however , they do not agree on how best to structure the reflective journal and if the format of the journal should vary for different student subgroups . first , journaling can be viewed as a transformative learning tool for the nursing instructor , challenging the attitudes and the practices of the learner . educators strive to awaken the novice to the perspective of the client , the family , the role of evidence , or the impact of the multifaceted health care system . students are encouraged to share the thoughts surrounding their actions and focus on the meaning of their experiences . the focus moves from accomplishing a list of behaviors correctly to the process of learning . through learning journaling can also be viewed as a skill requiring reasoning and writing expertise . with guidance and support , the clinical reasoning skills seen in the reflective journal can be further developed . in the epp review of the literature , tanner 's clinical judgment model lends structure to the clinical reasoning process of the nurse and the nursing student . reflection is guided using a consistent method , which is designed to promote competence of the baccalaureate nursing student . clinical reasoning is viewed as a skill , that is , structured , practiced , and ultimately greater proficiency is achieved . another aspect to consider is adding structure to the reflective journal , which is thought to promote reflection in nursing students . in an effort to promote learning and promote proficient reflection , nurse educators have developed models or guidelines to assist students to analyze their clinical experiences . guided reflection has been shown to move students past simply describing events toward more thorough introspection and concept synthesis regarding clinical experiences . by directing students to collect pertinent information , by probing and asking pointed questions , educators direct students toward thinking exercises that foster a deeper understanding . the process of reflection is guided rather than left open to free - form , unstructured comments . finally , the reflective journal should be a secure place that encourages the student to question and to elaborate on emotional responses or challenges to existing beliefs . fink defined optimal instructor feedback as being timely , caring , and discriminant . the lasater clinical judgment rubric describes the components of clinical decision making in a tool that structures the evaluative feedback from an instructor . as online educational systems develop , instructors have begun to use group discussion boards to postreflective journals to promote interactive group learning . there is a paucity of research on the use of online group discussion boards for reflective journaling . students collaborating on journals have demonstrated higher levels of reflection , suggesting benefit from a group process [ 12 , 13 ] . on the other hand , collaborative reflective journals have shown promise for some students and present a barrier to others as the journal requires expressing personal thoughts and receiving feedback in a setting that is not private . the research has shown that many students value reflective journals , but that is not consistent for all students . in one study , a group of students indicated that they were skeptical of the benefits of reflective journaling , the act of reflecting did not improve their practice , yet they recognized that there were positive impacts on practice for students who successfully reflected upon their practice . there have been no studies of student perception of the format of reflective journal or studies of student factors that may facilitate or hinder journaling effectiveness . no study has systematically analyzed the perceptions of students related to learning through reflective journals . what is the relative importance of each attribute involved in the journaling experience?what are changes that would increase satisfaction of students with the journaling experience?do differences in student characteristics lead to changes in their preferences ? do differences in student characteristics lead to changes in their preferences ? after receiving irb approval from university x , students in three undergraduate nursing classes were given the opportunity to voluntarily complete a confidential survey without an instructor present . the survey was offered to second semester junior students ( n = 41 ) , first semester senior students ( n = 40 ) , and final semester senior students ( n = 38 ) . within this convenience sample , 13 , 24 , and 19 completed surveys , respectively , were received from these classes for a response rate of 55.5% . delphi or expert panel of nurse educators convened to try and determine which attributes of reflective journaling would be viewed by students as the most important characteristics of the journaling process . the panel was comprised of four doctoral , and two masters prepared faculty members with 86 cumulative years of experience educating nursing students in the clinical setting . after extensive discussion and consideration of many possible components of reflective journaling , the panel determined that there were five major attributes contained within the student experience of reflective journaling . after determining these attributes the attributes and levels are described in table 1 . additionally , we asked four demographic questions to see if there were relationships between these characteristics and the preferences expressed for the five journaling attributes . these demographic variables are experience , sex , grade point average ( gpa ) , and primary language ( esl ) . gpa is divided into greater than 3.49 ( gpa 1 ) , between 2.5 and 3.5 ( gpa 2 ) , and less than 2.5 ( gpa 3 ) . esl is a self - reporting variable specifying whether english is their primary language , and students answer either yes ( esly ) or no ( esln ) . experience relates to the number of classes they have already completed where journaling was a component of the coursework . the students were either three trimesters from graduation ( exp 1 ) , two trimesters from graduation ( exp 2 ) , or in the final trimester before graduation ( exp 3 ) . in other words , the more coursework each student had completed , the greater exposure they had had to reflective journaling , as each trimester contains a journaling component in at least one course . conjoint analysis is a market - based research analysis model where potential and/or actual customers make choices based on their preferences . these choices are based on the individual attributes of the product under consideration and are considered jointly ( conjoint ) . for example , all else equal , most consumers would choose the cheapest computer . however , they can not have both of these attributes simultaneously because customers are forced to pay additional money for the faster computer should they desire more speed . people make tradeoffs every day as they try and balance the partial worth of many attributes like speed , color , size , power , convenience , durability , prestige , necessity , and price ( among others ) . a similar model exists in education . students , like computer purchasers , have preferences about what is important to them and are , in effect , buyers of nursing education . like the manufacturers of computers who care greatly about the preferences of their customers , the sellers of nursing education should also care about the preferences of their customers . for example , in reflective journaling , students might desire a short time to complete their journaling requirement and , at the same time , want a transformative experience which requires more time . each attribute and its corresponding levels were entered into the conjoint value analysis ( cva ) software program which automatically generates a survey . the survey began with the four demographic questions previously described and followed with twenty pair - wise discrete choice questions . each question presented the student respondent with a choice of two journaling experiences from which they had to choose one . ( scale 3 ) to indifferent ( scale 5 ) to somewhat prefer right each attribute is present in all choices , but the levels within each attribute are varied , and these variations are shuffled with each succeeding question . additionally , the cva questionnaire generator automatically moves attribute levels from left to right as the respondent proceeds through the questionnaire . a typical conjoint question is in table 2 , and the complete survey is available upon request . after data entry , the cva software runs multiple ordinary least squares ( olss ) functions by performing simultaneous regressions of the five independent attribute variables on the dependent variable of preference . for a detailed explanation of cva the results are presented as average utilities ( preferences ) , and the average importance of each attribute is estimated , and an r is calculated . the results are then exported to the sawtooth marketing simulation ( smrt ) software which runs a series of simulation algorithms where the different levels of the same attribute are compared to each other while holding all other attributes the same . these results estimate the share preferences for each attribute level relative to other levels . through multiple iterations , all preferences for each level are estimated in relationship to each other by simply dividing one into the next . finally , to estimate the changes ( if any ) that the demographic variables have on the preferences of attributes levels within reflective journaling , the marketing simulation software takes the attribute preference data and filters it through the four demographic questions and provides a result containing product shares of preference and standard errors . through these two statistics , confidence intervals and p values ( z scores ) the importance ( table 3 ) of the five attributes varies from a low of 16.75% ( format ) to a high of 23.58% ( time ) . the lack of any one attribute being extremely high or low relative to the other attributes suggests that all five attributes were considered and paid attention to by the respondents . this is further reinforced by an r squared ( see table 4 ) of 0.777 . that is , this model explains 77.7% of the variability of journaling preferences among nursing students and is clearly superior to a random predictor of this variation . the utilities ( see table 4 ) are interval - scaled values without a real zero . therefore , utilities represent weights relative to each other , and the sum of the three levels equals zero . it is clear that students prefer a shorter time , complete confidentiality , recognition of their behavior , one - time complete feedback , and a semistructured format . however , because these are interval values , they provide preferences only . for these data to be useful in the real world of developing courses and , more specifically , journaling experiences , there must be a real zero . additionally , to accurately compare different levels of any single attribute , all other attributes must be held constant . the smrt uses a randomized first choice simulation algorithm to accomplish this . for a detailed explanation of the smrt software package , please refer to the sawtooth research paper on market simulations referenced in the bibliography . students prefer a weekly time commitment of 15 minutes over 30 minutes ( p < 0.05 ) , and they prefer 30 minutes over 45 minutes ( p < 0.05 ) . by decreasing the time commitment from 45 to 30 minutes , student satisfaction increases by 15% and if students are currently spending 45 minutes a week and an instructor reduces that to 15 minutes ( p < 0.05 ) , student satisfaction increases by 54% . ( nonanonymous ) option were rejected by students . by moving the journaling experience from open to either completely confidential ( p < 0.05 ) or to shared content ( p < 0.05 ) however , it is less clear whether students have a problem sharing their thoughts anonymously . however , by taking the square root of the squared sum of the two separate standard error terms and dividing that into the difference of the partial shares , a p value is easily found in a z table ( 0.07 ) . therefore , students also prefer complete confidentiality to anonymous sharing ( p = 0.07 ) , but their satisfaction only increases by 9% by moving from the latter to the former . students want results from their journaling efforts though they are relatively indifferent whether they simply learn to recognize their behaviors or have those behaviors transformed . in both iterations in fact , by changing the journaling experience from one where the students perceive that no change has occurred to one where they either recognize their behaviors ( p < 0.05 ) or have them transformed ( p < 0.05 ) , student satisfaction increases by 41% and 38% , respectively . however , the two 95% c.i . that compared recognition to transformation almost completely overlap . so although instructors may think that it is a higher level of learning for students to be transformed versus simply recognizing positive and negative behaviors , students do not value that difference . students value feedback over simply checking their work for completeness although they are indifferent whether that feedback comes only once or multiple times . by moving from a model of checking for completeness to one where students receive feedback once ( p < 0.05 ) , student satisfaction increases by 11% . however , the 95% intervals for checking for completeness versus multiple feedbacks have overlapping tails and the p value ( 0.06 ) is calculated ( as above ) using the z table . finally , students are indifferent to a single feedback or multiple feedbacks . the 95% c.i . for both of these levels almost completely overlap , and there is no difference . students do not value a completely structured format . by moving from a completely structured format to either a semi - structured ( p < 0.05 ) or open format ( p = 0.06 ) student satisfaction increases by 10% and 9% , respectively . however , students did not perceive much difference between the semi - structured or open format . there was no difference . finally , to estimate the changes ( if any ) that the demographic variables have on the preferences of attributes levels within reflective journaling , the attribute preference data was filtered through the four demographic questions . most of the demographic categories had no significant effect on student preferences , and these are not reported though the data is available upon request . the sex and gpa of the respondents had no significant effect on student preferences for journaling attributes . that is , all calculated confidence intervals that compared these two demographic variables had a great deal of overlap . the experience level of the students did have an effect on the value of time and the preference of format . those students with less experience ( exp 1 ) had a much greater preference for less time spent journaling ( p < 0.023 ) and a much greater preference for a structured format ( p < 0.39 ) . conversely , those students with the most experience ( exp 3 ) had a much greater preference for a free - form format ( p < 0.39 ) when compared to students with less journaling experience . students that self - reported english as their second language ( esly ) viewed the results of journaling differently . that is , although the no effect level of result was perceived negatively by all students , it was perceived much more negatively by non - esl students . those students whose first language was english ( esln ) preferred feedback to no feedback , and esl students did not . the results for the esl demographic should be viewed with some caution as there were only 2 students reporting themselves as esl . although the weakness of a small cohort is captured in the standard error calculation , which leads directly to the p value , it still gives pause . we report those results here because mathematically the differences were significant but will leave it up to the reader to decide . question 1 : what is the relative importance of each of the five attributes involved in the journaling experience ? in descending order , the most important characteristics of journaling are time , confidentiality , results , feedback and format ( table 1 ) . question 2 : what are the changes that would most increase the satisfaction of students with the journaling experience ? to increase satisfaction for students , instructors should strive to reduce the time students spend journaling , maintain anonymity ( though content might be shared ) , and instill a perception in students that reflective journaling will have a tangible effect on each student 's ability to transform or simply recognize their behaviors ( table 5 , far right column ) . question 3 : do differences in student characteristics lead to changes in their preferences about the journaling experience . however , students with less experience in journaling value time more than the entire cohort and students with more experience prefer an open format with fewer instructor cues . additionally , esl students placed less value on instructor feedback and behavior recognition or transformation . however , because of the small sample size , these results should be viewed with some caution ( table 6 ) . the delphi panel included this dimension as an attribute of the journal recognizing that it is a paramount concern of nursing students . nursing instructors will probably read this and recognize the challenge of creating journaling exercises that are both time efficient and produce the requisite level of reflection . however , students would highly value new journaling exercises that prove to be more time efficient . online , discussion systems should be developed that include areas for confidential discussion and individual feedback , as well as open discussions areas where students can benefit from interaction and collaboration . in the age of the electronic classroom , anonymity and collaboration it is interesting to note the undergraduate student 's comments on the structured journal considering the development of various models or guidelines [ 7 , 8 ] . students preferred either a semi - structured or an open , free - form journal . educators working on models that structure student responses should be advised to insure that exercises include areas for students to write freely without predetermined structure . there is evidence that using a model or structured journal early in a nursing program and moving to a predominantly unstructured journal later in the program will be valued more highly by students . instructors should insure that whether using a model or not , students value substantive feedback after they have prepared reflective journals . esl students viewed journaling differently compared to non - esl students . c. kuo developed an online writing system to support nonnative students with writing exercises . the system offers a supportive environment encouraging writing practice , peer review , and use of an e - portfolio , online dictionary , phrase lists , and so forth . research findings are beginning to reveal more about the kinds of support that will benefit this group of students . therefore , there exists a risk that a significant variable was overlooked by the delphi panel and not included in the survey . the omission of significant variables can lead to specification error which has the potential to bias coefficients and results . the students in our sample attend the same school , are taught by the same teachers , and live in the state of x. journaling experiences nationwide or worldwide might be quite different than those experienced by the students at our institution and , therefore , these results might not generalize to other populations in other institutions across the country . for example , there were only 2 esl students , 6 male students , and 3 who reported a gpa under 2.5 . all calculations in this study make use of the standard error term , and standard errors take into account small sample sizes . however , we would still view all of the small sample conclusions with some caution . similarly , regardless of sample size , a p value of 0.05 still means that we are only correct 19 out of 20 times and , therefore , could be wrong . marketing professionals have surveyed their customers and potential customers in an effort to provide the product or service that is most desired by them . we also wanted to find out what aspects of journaling our customers ( students ) value . this study applied the statistical method of conjoint value analysis , a sound quantitative modeling method that has been used in the field of marketing for many years . results indicated that all students value time , confidentiality , results , feedback , and format , in that order . the less - experienced students valued a structured journal , while the more advanced students valued a more free - form journal . nurse educators should consider these attributes when developing and refining the journals in their courses . while journaling is a process that supports learning , it is also very personal . students must see that the journaling system is confidential and that they will receive thoughtful feedback from the instructors . this study helps us better understand nursing students , our customers , and to better design journaling experiences that future students might find more satisfying . more research is needed to test new journaling systems as well as to explore the unique needs of student subgroups , particularly esl students .

due to their unique structural and functional properties , subplate cells are ideally suited to function as important amplifying units within the developing neocortical circuit . subplate neurons have extensive dendritic and axonal ramifications and relatively mature functional properties , i.e. their action potential firing can exceed frequencies of 40 hz . at earliest stages of corticogenesis subplate cells receive functional synaptic inputs from the thalamus and from other cortical and non - cortical sources . glutamatergic and depolarizing gabaergic inputs arise from cortical neurons and neuromodulatory inputs arise from the basal forebrain and other sources . activation of postsynaptic metabotropic receptors , i.e. muscarinic receptors , elicits in subplate neurons oscillatory burst discharges which are transmitted via electrical and chemical synapses to neighbouring subplate cells and to immature neurons in the cortical plate . the tonic non - synaptic release of gaba from gabaergic subplate cells facilitates the generation of burst discharges . these cellular bursts are amplified by prominent gap junction coupling in the subplate and cortical plate , thereby eliciting 1020 hz oscillations in a local columnar network . thus , we propose that neuronal networks are organized at earliest stages in a gap junction coupled columnar syncytium . we postulate that the subplate does not only serve as a transient relay station for afferent inputs , but rather as an active element amplifying the afferent and intracortical activity . this study used a statistical technique , conjoint value analysis , to determine student perceptions related to the importance of predetermined reflective journaling attributes . an expert delphi panel determined these attributes and integrated them into a survey which presented students with multiple journaling experiences from which they had to choose . after obtaining irb approval , a convenience sample of 66 baccalaureate nursing students completed the survey . the relative importance of the attributes varied from a low of 16.75% ( format ) to a high of 23.58% ( time ) . the model explained 77% of the variability of student journaling preferences ( r2 = 0.77 ) . students preferred shorter time , complete confidentiality , one - time complete feedback , semistructured format , and behavior recognition . students with more experience had a much greater preference for a free - form format ( p < .05 ) when compared to students with less journaling experience . additionally , the results of english as a second language students were significantly different from the rest of the sample . in order to better serve them , educators must consider the relative importance of these attributes when developing journaling experiences for their students .

acute pancreatitis is an inflammatory disease characterized by the presence of acute abdominal pain and/or increased levels of serum pancreatic enzymes . more importantly , takeda et al . have indicated that the mortality rate was of approximately 20% when acute pancreatitis developed to severe acute pancreatitis ( sap ) . similarly , several lines of evidence have demonstrated that systemic inflammatory response syndrome ( sirs ) and multiple - organ dysfunction syndrome ( mods ) are the 2 most common causes of death in the early stage ( within approximately 2 weeks ) of patients with sap . previous research has suggested that acute respiratory distress syndrome ( ards ) , which is a clinical manifestation of lung injury , is one of the earliest manifestations of functional deterioration in human organs and is one of the most common and serious complications of sap . the most severe clinical manifestations of ards exhibit significant hypoxemia with bilateral pulmonary infiltrates consistent with edema . currently , the criteria for the diagnosis of ards are mainly based on the berlin definition , which includes the original diagnosis of acute lung injury ( ali ) and ards : acute onset of respiratory distress , hypoxemia ( ali : pao2/fio2 300 mmhg , ards : pao2/fio2 200 mmhg ) , bilateral consolidation of chest radiograph , and absence of clinical findings of cardiogenic pulmonary edema . in practice , the complexity of multiple - item criteria of ards often causes inconvenience for physicians in the management of patients with sap . if ards also causes changes in several related bio - markers , similar to the characteristic elevation of troponin in myocardial infarction , this bio - marker might be useful to help diagnose of sap - induced ards . previous reports indicate that pulmonary surfactant consists principally of various lipids and proteins ( surfactant proteins ) . furthermore , the major constituent of surfactant proteins is sp - a , and the remainder includes sp - b , sp - c , and sp - d . recently , a study suggested that sp - a is a large multimeric protein found in the airways and alveoli . several lines of evidence have shown that sp - a not only mediates the inflammatory response , but also controls vascular permeability , which are considered to be important factors in the development of ards . a large number of studies have shown that sp - a levels in bronchoalveolar lavage fluid ( balf ) are significantly lower in patients with bleomycin - induced ards compared with healthy volunteers . in contrast , the serum levels of sp - a were raised in patients with bleomycin - induced ards . thus , the serum levels of sp - a may be suggested as a sensitive and specific serum marker on the diagnosis of sap - induced ards . it has been suggested that the presence of qualitatively assessed ( yes or no ) diffuse alveolar damage ( dad ) should be considered the criterion standard for the diagnosis of ards . to improve the early detection and diagnosis of sap - induced ards , determination of serum levels of sp - a in sap - induced ards rats was used . in the present study , we therefore evaluated serum sp - a levels according to the criterion standard for the diagnosis of ards to evaluate the qualitative assessment of presence of diffuse alveolar damage ( dad ) in the diagnosis of sap - induced ards in an animal model . one hundred male wistar rats weighing 250300 g were purchased from cavens laboratory animal company , changzhou , jiangsu , china . six rats were housed per cage with food and water available ad libitum and maintained on a 12-h light / dark cycle ( lights on at 07:00 am ) . eighty rats were randomly divided into 2 groups ( n=40 each ) : the sham - operated ( so group ) and the severe acute pancreatitis ( sap group ) and then randomly subdivided into 1-h , 2-h , 3-h , and 4-h subgroups , with 10 rats in each subgroup . the rats in the 1-h , 2-h , 3-h , and 4-h subgroups of the sap group , a total of 40 rats , were subdivided into a sap - induced ards group ( ards group ) and a sap without ards group ( non - ards group ) according to the diagnosis of ards for lung tissue , including the presence of dad , qualitatively assessed ( yes or no ) after the operation . the other 20 rats were regarded as the baseline and euthanized without any operation in initial experiments . intra - peritoneal injection of 1% sodium pentobarbital ( 50 mg / kg ; sigma , st . louis , mo , usa ) was used for abdominal cavity anesthesia to all rats of the 1-h , 2-h , 3-h , 4-h subgroups . animal care was in accordance with the national institutes of health guide for the care and use of laboratory animals , and was approved by the animal use and protection committee of soochow university . the abdominal surgery of the so group ( n=40 ) consisted of opening the abdomen , flipping the pancreas , returning them to their original position , then closing the abdominal cavity with 2 layers of sutures . louis , mo , usa ) was administered into the common biliopancreatic duct of the sap group ( n=40 ) by retrograde injection . we identified the duodenal papilla inside the duodenum duct wall , and then used a no . a segmental epidural catheter was inserted into the duodenum cavity through the hole , and inserted retrogradely into the biliary - pancreatic duct through the papilla . this was followed by retrograde transfusion of 4.5% sodium taurocholate solution ( 4.5% ; 0.1 ml/100 g ) by a microinjection pump at 0.1 ml / min . all rats of the 2 groups were injected with normal saline ( 20 ml / kg ) through the vena caudalis to compensate for loss of fluid during surgery . the rats in all groups were euthanized at 1 , 2 , 3 and 4 hours , according to the subgroup , after the operation . blood samples ( 5 ml ) were collected from the heart , and were mixed with edta anticoagulative liquor , then were centrifuged at 3000 sera of their blood samples were collected and maintained at 4c for the detection of sp - a . serum levels of sp - a were determined by the elisa method by using the rat sp - a elisa kit purchased from mybiosource ( san diego , california , usa ) . the antigens were measured by elisa inhibition assays using antibodies raised against alveolar proteinosis - derived sp - a . briefly , in order to free the sp - a from any associated plasma or surfactant components , aliquots were first treated with edta , sds , and triton x-100 . serial dilutions of the samples in pbst containing 0.25% bsa ( w / v ) were incubated in an elisa plate with aliquots of the respective antibody . free antibody was captured using a second elisa plate coated with purified sp - a ( 1 g / ml ) and the amount measured using alkaline phosphatase conjugated igg against rabbit immunoglobulins and 15 mm disodium p - nitrophenyl phosphate in 1.0 m diethanolamine and 0.5 mm mgcl 2 as a substrate . after 1 h , the absorbance of the substrate was measured at 405 nm using a dynatech mr5000 reader ( dynatech laboratories , chantilly , va , usa ) . the assayzap program ( biosoft , ferguson , mo , usa ) was used to generate a standard curve and to compute the concentration of sp - a in each sample . standards , assayed in quadruplicate , were included in each elisa plate at 8 serial dilutions ( range 1.95250 ng / ml for sp - a , r>0.99 ) . the antibodies used do not react with any other known antigens and the assays have coefficients of variance of ~6% . rat pancreatic and lung tissues were collected , fixed in 10% formalin , embedded in paraffin , and sectioned into 4- sections , after which they were stained by hematoxylin and eosin ( h&e ) staining for histopathological observation . histopathology of the pancreas and lungs was scored and classified by 2 professional pathologists using a double - blind method . each group randomly selected 3 slices ; for each slice , 10 high - power fields of vision were again randomly selected , and finally the extent of pancreatic tissue damage by edema , infection , hemorrhage , and necrosis were evaluated . the pathological score for pancreatic tissue was calculated according to rongiones standards as a reference , with a minimum score of 0 and the highest score of 4 . the pathological grade for lung tissue was according to lei as a reference , with a minimum grade of 0 and the highest grade of iii . criteria for the diagnosis of ards for lung tissue included the presence of diffuse alveolar damage ( dad ) , qualitatively assessed ( yes / positive result or no / negative result ) as : intra - alveolar edema , alveolar type i cell necrosis , alveolar type ii cell ( cuboidal cells ) proliferation progressively covering the denuded alveolar - capillary membrane , interstitial proliferation of fibroblasts and myofibroblasts , or organizing interstitial fibrosis . values of sp - a for ards were determined by the receiver operating characteristic curve ( roc ) . the comparison among multiple groups was analyzed with 1-dimensional analysis of variance after data conversion . ards group and so group + non - ards group was tested with nonparametric rank sum test . true- positive ( tp ) was defined as sp - a values higher than the cut - off value in rats with sap induced - ards groups ( ards group ) . false - negative ( fn ) included rats with ards but who scored negative . sensitivity and specificity formulas for calculation were : sensitivity = tp/(tp+fn)100% , specificity = tn/(tn+fp)100% , diagnostic accuracy=(tp+tn)/(tp+fp+tn+fn)100% , positive predictive value = tp/(tp+fp ) 100% , and negative predictive value = tn/(tn+fn)100% . one hundred male wistar rats weighing 250300 g were purchased from cavens laboratory animal company , changzhou , jiangsu , china . six rats were housed per cage with food and water available ad libitum and maintained on a 12-h light / dark cycle ( lights on at 07:00 am ) . eighty rats were randomly divided into 2 groups ( n=40 each ) : the sham - operated ( so group ) and the severe acute pancreatitis ( sap group ) and then randomly subdivided into 1-h , 2-h , 3-h , and 4-h subgroups , with 10 rats in each subgroup . the rats in the 1-h , 2-h , 3-h , and 4-h subgroups of the sap group , a total of 40 rats , were subdivided into a sap - induced ards group ( ards group ) and a sap without ards group ( non - ards group ) according to the diagnosis of ards for lung tissue , including the presence of dad , qualitatively assessed ( yes or no ) after the operation . the other 20 rats were regarded as the baseline and euthanized without any operation in initial experiments . intra - peritoneal injection of 1% sodium pentobarbital ( 50 mg / kg ; sigma , st . louis , mo , usa ) was used for abdominal cavity anesthesia to all rats of the 1-h , 2-h , 3-h , 4-h subgroups . animal care was in accordance with the national institutes of health guide for the care and use of laboratory animals , and was approved by the animal use and protection committee of soochow university . the abdominal surgery of the so group ( n=40 ) consisted of opening the abdomen , flipping the pancreas , returning them to their original position , then closing the abdominal cavity with 2 layers of sutures . louis , mo , usa ) was administered into the common biliopancreatic duct of the sap group ( n=40 ) by retrograde injection . we identified the duodenal papilla inside the duodenum duct wall , and then used a no . a segmental epidural catheter was inserted into the duodenum cavity through the hole , and inserted retrogradely into the biliary - pancreatic duct through the papilla . this was followed by retrograde transfusion of 4.5% sodium taurocholate solution ( 4.5% ; 0.1 ml/100 g ) by a microinjection pump at 0.1 ml / min . all rats of the 2 groups were injected with normal saline ( 20 ml / kg ) through the vena caudalis to compensate for loss of fluid during surgery . the rats in all groups were euthanized at 1 , 2 , 3 and 4 hours , according to the subgroup , after the operation . blood samples ( 5 ml ) were collected from the heart , and were mixed with edta anticoagulative liquor , then were centrifuged at 3000 sera of their blood samples were collected and maintained at 4c for the detection of sp - a . serum levels of sp - a were determined by the elisa method by using the rat sp - a elisa kit purchased from mybiosource ( san diego , california , usa ) . the antigens were measured by elisa inhibition assays using antibodies raised against alveolar proteinosis - derived sp - a . briefly , in order to free the sp - a from any associated plasma or surfactant components , aliquots were first treated with edta , sds , and triton x-100 . serial dilutions of the samples in pbst containing 0.25% bsa ( w / v ) were incubated in an elisa plate with aliquots of the respective antibody . free antibody was captured using a second elisa plate coated with purified sp - a ( 1 g / ml ) and the amount measured using alkaline phosphatase conjugated igg against rabbit immunoglobulins and 15 mm disodium p - nitrophenyl phosphate in 1.0 m diethanolamine and 0.5 mm mgcl 2 as a substrate . after 1 h , the absorbance of the substrate was measured at 405 nm using a dynatech mr5000 reader ( dynatech laboratories , chantilly , va , usa ) . the assayzap program ( biosoft , ferguson , mo , usa ) was used to generate a standard curve and to compute the concentration of sp - a in each sample . standards , assayed in quadruplicate , were included in each elisa plate at 8 serial dilutions ( range 1.95250 ng / ml for sp - a , r>0.99 ) . the antibodies used do not react with any other known antigens and the assays have coefficients of variance of ~6% . rat pancreatic and lung tissues were collected , fixed in 10% formalin , embedded in paraffin , and sectioned into 4- sections , after which they were stained by hematoxylin and eosin ( h&e ) staining for histopathological observation . histopathology of the pancreas and lungs was scored and classified by 2 professional pathologists using a double - blind method . each group randomly selected 3 slices ; for each slice , 10 high - power fields of vision were again randomly selected , and finally the extent of pancreatic tissue damage by edema , infection , hemorrhage , and necrosis were evaluated . the pathological score for pancreatic tissue was calculated according to rongiones standards as a reference , with a minimum score of 0 and the highest score of 4 . the pathological grade for lung tissue was according to lei as a reference , with a minimum grade of 0 and the highest grade of iii . criteria for the diagnosis of ards for lung tissue included the presence of diffuse alveolar damage ( dad ) , qualitatively assessed ( yes / positive result or no / negative result ) as : intra - alveolar edema , alveolar type i cell necrosis , alveolar type ii cell ( cuboidal cells ) proliferation progressively covering the denuded alveolar - capillary membrane , interstitial proliferation of fibroblasts and myofibroblasts , or organizing interstitial fibrosis . values of sp - a for ards were determined by the receiver operating characteristic curve ( roc ) . the comparison among multiple groups was analyzed with 1-dimensional analysis of variance after data conversion . ards group and so group + non - ards group was tested with nonparametric rank sum test . true- positive ( tp ) was defined as sp - a values higher than the cut - off value in rats with sap induced - ards groups ( ards group ) . false - negative ( fn ) included rats with ards but who scored negative . sensitivity and specificity formulas for calculation were : sensitivity = tp/(tp+fn)100% , specificity = tn/(tn+fp)100% , diagnostic accuracy=(tp+tn)/(tp+fp+tn+fn)100% , positive predictive value = tp/(tp+fp ) 100% , and negative predictive value = tn/(tn+fn)100% . the glandular architecture of the pancreas in the so group was entirely normal ( h&e ) ( figure 1 ) . the architecture of the lung in the so group was entirely normal ( h&e ) ( figure 2 ) . histopathologic features of the pancreas in the sap group included various degrees of edema , minimal necrosis , and inflammatory infiltration ( h&e ) ( figure 3 ) . according to the criteria for the diagnosis of ards , the presence of dad was qualitatively assessed ( yes or no ) ; in the 1-h , 2-h , 3-h , and 4-h subgroups we found 1 , 5 , 9 , and 10 cases of ards , respectively . therefore , the sap group was then subdivided into the ards group ( 25 rats ) and non - ards group ( 15 rats ) according to the criteria for the diagnosis of ards . histopathologic features of lung in the ards group included the presence of dad , intra - alveolar edema , and organizing interstitial fibrosis ( h&e ) ( figure 4 ) . serum sp - a levels ( means sd ) in the baseline , so , and sap groups were 43.1514.29 , 51.9116.99 , and 193.435.37 . there was no statistical difference between so group and baseline in serum sp - a levels ( p>0.05 ) . serum sp - a levels of the sap group were significantly increased compared to baseline and so group ( * p<0.05 , p<0.05 ) ( figure 5 ) . serum sp - a levels ( means sd ) in the so group , ards group , and non - ards group were 51.9116.99 , 198.029.73 , and 185.743.21 ug / ml , respectively . serum sp - a levels were significantly increased from the ards group to the so group ( * p<0.05 ) and from the non - ards group to the so group ( p<0.05 ) . there was no statistical difference between the ards group and non - ards group in serum sp - a levels ( p>0.05 ) ( figure 6 ) . according to the analysis of roc curves , the best cut - off value for the serum sp - a level for the diagnosis of sap - induced ards was 150 ug / ml . in analysis of all sap - induced ards rats , the area under the roc curve of sp - a was 0.88 ( figure 7 ) . the sensitivity , specificity , positive predictive value , negative predictive value , and accuracy of sp - a in the diagnosis of sap - induced ards were 100.0% , 81.8% , 71.4% , 100.0% , and 87.5% , respectively ( table 1 ) . the glandular architecture of the pancreas in the so group was entirely normal ( h&e ) ( figure 1 ) . the architecture of the lung in the so group was entirely normal ( h&e ) ( figure 2 ) . histopathologic features of the pancreas in the sap group included various degrees of edema , minimal necrosis , and inflammatory infiltration ( h&e ) ( figure 3 ) . according to the criteria for the diagnosis of ards , the presence of dad was qualitatively assessed ( yes or no ) ; in the 1-h , 2-h , 3-h , and 4-h subgroups we found 1 , 5 , 9 , and 10 cases of ards , respectively . therefore , the sap group was then subdivided into the ards group ( 25 rats ) and non - ards group ( 15 rats ) according to the criteria for the diagnosis of ards . histopathologic features of lung in the ards group included the presence of dad , intra - alveolar edema , and organizing interstitial fibrosis ( h&e ) ( figure 4 ) . serum sp - a levels ( means sd ) in the baseline , so , and sap groups were 43.1514.29 , 51.9116.99 , and 193.435.37 ug / ml , respectively . there was no statistical difference between so group and baseline in serum sp - a levels ( p>0.05 ) . serum sp - a levels of the sap group were significantly increased compared to baseline and so group ( * p<0.05 , p<0.05 ) ( figure 5 ) . serum sp - a levels ( means sd ) in the so group , ards group , and non - ards group were 51.9116.99 , 198.029.73 , and 185.743.21 ug / ml , respectively . serum sp - a levels were significantly increased from the ards group to the so group ( * p<0.05 ) and from the non - ards group to the so group ( p<0.05 ) . there was no statistical difference between the ards group and non - ards group in serum sp - a levels ( p>0.05 ) ( figure 6 ) . according to the analysis of roc curves , the best cut - off value for the serum sp - a level for the diagnosis of sap - induced ards was 150 ug / ml . in analysis of all sap - induced ards rats , the area under the roc curve of sp - a was 0.88 ( figure 7 ) . the sensitivity , specificity , positive predictive value , negative predictive value , and accuracy of sp - a in the diagnosis of sap - induced ards were 100.0% , 81.8% , 71.4% , 100.0% , and 87.5% , respectively ( table 1 ) . in the present study , we evaluated serum sp - a levels for the diagnosis of sap - induced ards in a rat model . importantly , the sap - induced ards rat model is mostly suitable for investigating the effectiveness and specificity of a particular therapeutic approach . the model that we chose had to be similar to the etiology and convenient for the experimental operations . some models focus on etiology , such as the duct ligation model and the taurocholate - induced pancreatitis model . but the latter is more simple , and less damaging to the animals , which mimic biliary pancreatitis in the clinical setting better than the duct ligation model . several lines of evidence have indicated that the taurocholate - induced pancreatitis model induced by ards was successfully 34 h after the operation . thus , we constructed this model by using sodium taurocholate solution injected into the common biliopancreatic duct . according to the criteria for the diagnosis of ards , 1 , 5 , 9 , and 10 cases of ards were determined as positive results in the 1-h , 2-h , 3-h , and 4-h subgroups , respectively . the results of the present study are consistent with previous findings that sap leading to a systemic inflammatory response was characterized by widespread leukocyte activation and distant organ injury ( e.g. , ards ) [ 2628 ] . our previous study showed that a definite correlation exists between pathological changes ( pancreas and lung ) and progression of sap . in the present study , we found that the glandular architecture of the pancreas and the architecture of the lung in the so group were entirely normal ( h&e ) ( figures 1 and 2 ) . furthermore , histopathologic features of the pancreas , various degrees of edema , minimal necrosis , and inflammatory infiltration ( h&e ) were detected in glandular architecture of the pancreas in the sap group ( figure 3 ) . the sap group was subdivided into the ards group ( 25 rats ) and non - ards group ( 15 rats ) according to criteria including presence of dad , intra - alveolar edema , and organizing interstitial fibrosis ( h&e ) ( figure 4 ) . these results are similar to findings of the present study , suggested that the cases of positive results , which included the presence of dad , was qualitatively assessed ( yes or no ) , showing an increasing trend associated with longer time in the preceding 4 h in the rat model of sap - induced ards . this gap in serum levels of sp - a has previously been reported in many studies , with the results being similar to those in the present study baseline , 43.1514.29 ug / ml vs. so group 51.9116.99 ug / ml vs. sap group 193.435.37 ug / ml ( figure 5 ) ; so group , 51.9116.99 ug / ml vs. ards group 198.029.73 ug / ml vs. non - ards group 185.743.21 ug / ml ( figure 6 ) . these findings demonstrated that ards , the most common and serious complications of sap , was induced in the sap group and contributed to an elevation of sp - a in serum in this study . however , the underlying mechanism has not yet been fully elucidated and may be related to multiple factors . we propose that the possible mechanisms include epithelial injury and leak increased secretion of sp - a per type ii cell , increased leakage from the airspace to the interstitium , an increase in total number of type ii cells per lung due to diffuse hyperplasia , and decreased clearance from the vascular compartment . in addition , the degree of lung injury and sp - a levels leaked from the airspace to the serum was similar in the ards group and non - ards group . moreover , the presence of dad was not observed in the non - ards group because of multiple factors , such as the time of lung injury , individual differences , and the degree of lung injury , rather than the absence of lung injury . found that serum sp - a levels are upregulated by an acute inflammatory stress ; therefore , we used determination of serum levels of sp - a to diagnose sap - induced ards in the rat model . our data analysis showed that sp - a>150 ug / ml statistically discriminates between sap - induced ards and other groups ( so group and non - ards group ) with 100.0% sensitivity and 81.8% specificity . at this cut - off value , the diagnostic accuracy was the highest , and the area under the roc curve of sp - a was 0.88 ( figure 7 ) . furthermore , sp - a>150 ug / ml was diagnosed as sap - induced ards with 71.4% positive predictive value , 100.0% negative predictive value , and 87.5% accuracy in this study . these data demonstrated that the accuracy of diagnosis of sp - a for sap - induced ards is relatively high . therefore , the sp - a , as lung - specific bioactive surface proteins , are valuable parameters for the prediction of sap - induced ards . interestingly , because it is impossible to collect the human pancreatic and lung tissues in the clinical course of treatment , there is little data on this value . we , therefore consider that our findings can be extrapolated to other animals and to humans . although further clinical studies are required to confirm this , rats with sp - a greater than 150 ug / ml and high pulmonary vascular permeability index may be typical for sap - induced ards . in addition , ellsa can detect small changes in sp - a content and serum sp - a level can be easily , quickly , and repeatedly measured at the bedside . serum sp - a level correlates with the progression of lung injury and predicts progression to ards in patients with increased risk . thus , serum sp - a was chosen in this study as the first validated quantitative measure for sap - induced ards and we believe that monitoring serum sp - a level in peripheral blood of sap patients is helpful to evaluate whether sap induces ards . the serum levels of sp - a may be useful as a sensitive and specific serum marker for the diagnosis of sap - induced ards , as confirmed in the present study . however , a great limitation of the present study is that we did not observe the long - term clinical significance , and future studies are required to investigate this . mucormycosis , also known as zygomycosis , is an opportunistic and lethal mycological infection caused by a fungus of the order mucorales . it is mostly observed in immunocompromised patients such as those with hematological malignancy or those who have solid organ transplantation ( sot ) or bone marrow transplants . the infection usually starts in the middle nasal meatus and spreads to the paranasal sinuses , the orbit and the intracranial structures by direct extension or through blood vessels . early diagnosis , initiating prompt surgical and medical therapy and correcting the underlying conditions play a vital role in eliminating this infection , but mortality remains high . mucormycosis is uncommon in sot recipients and most cases have been reported after liver or renal transplantation . a 52-year - old male patient was transferred to our department from another center for retroorbital pain , headache and decreased vision accuracy in the left eye for the past 36 h. five months earlier , he had undergone orthotopic heart transplantation under the bicaval technique due to ischemic heart failure . his medical history was significant for type ii diabetes mellitus , poorly controlled by insulin . he also had hypertension and dyslipidemia ( both under treatment ) and nondialysis - dependent chronic kidney disease . his baseline immunosuppressive regimen consisted of prednisone , tacrolimus and mycophenolic acid and he was also taking prophylactic antibiotics : cotrimoxazole , valganciclovir and isoniazid with pyridoxine . physical examination on admission showed that the patient was alert , orientated , afebrile and his vital signs were stable . anterior rhinoscopy , endoscopy with 0 rigid endoscope and examination of the oral cavity were unremarkable . although the nasal exploration was normal , in view of the symptoms and the previous transplant , two samples endoscopically directed , one of the nasal fossa by swabbing and the second of the middle meatus were collected . blood tests showed glucose levels of 248 mg / dl ( 13.8 mmol / l ) and creatinine of 3.63 mg / dl . computed tomography ( ct ) scan revealed soft tissue opacification of left anterior ethmoid sinus and inflammatory changes in the apex of the left orbit [ figure 1a ] . ( a ) computed tomography scan showed soft tissue opacification of left ethmoid sinus ( white arrows ) and inflammatory changes in the apex of the left orbit ( black arrows ) . ( b ) fundus of the left eye with edema and papillary pallor ( * ) , posterior pole retinal edema ( white arrows ) and arteriolar sclerosis ( black arrows ) . ( c ) left nasal endoscopy with 0 rigid endoscope , showing black necrotic eschar ( white arrows ) through the left middle meatus and the middle turbinate ( black arrows ) . nasal septum ( * ) , ( d ) lactophenol cotton blue culture showing rhizopus fungus . sporangiophore ( black arrow ) ( lactophenol cotton blue stain , x1000 ) the metabolic derangement was treated , intravenous antibiotics ( piperacillin / tazobactam 2000/250 mg 8 hourly ) was started and insulin was administered on a sliding scale . on the second day of hospitalization , the patient 's clinical status worsened dramatically . left eye movements were limited in all directions ( frozen eye ) and the left pupil was mydriatic . signs of central retinal artery occlusion were found on the left fundoscopy : edema and papillary pallor , posterior pole retinal edema and arteriolar sclerosis [ figure 1b ] . a second nasal 0 rigid endoscope showed necrotic tissue in the left middle meatus and the middle turbinate [ figure 1c ] . the lactophenol cotton blue preparation revealed a sporangium located on the columella at the apical end of the sporangiophore [ figure 1d ] . following the confirmation of rm , intravenous liposomal amphotericin b the magnetic resonance imaging showed that infection had progressed in the apex of the left orbit , involving the cavernous sinus . we proposed extensive surgical treatment via left paralateral rhinotomy with orbital exenteration and total ethmoidectomy . its incidence has increased over the last 20 years due to increasing use of immunosuppressive drugs . it appears most frequently in the first 6 months after transplantation due to the higher immunosuppression . the incidence ranges from 0.4% to 16% depending on the sot type and is 0%0.6% in heart transplant recipients . other significant predisposing conditions to mucormycosis in sot recipients are poorly controlled diabetes mellitus and renal failure . the fungus spreads quickly in a medium of elevated glucose and acid ph and it is unusual in patients with metabolically controlled diabetes . the main protection against hyphae is neutrophils , but low serum ph decreases their pathogenic and chemotactic capacity . our patient had three predisposing conditions : immunosuppression for sot , poorly controlled diabetes mellitus and renal failure . three main ways of transmission of mucormycosis in transplant recipients have been reported : inhalation in approximately 70% , ingestion in 2% and percutaneous introduction in 13% . it then extends to the orbit and periorbital area where it can produce the orbital apex syndrome with ophthalmoplegia , ptosis , mydriasis and decreased visual accuracy . finally , it disseminates to the cavernous sinus and then to the meninges and brain . clinical presentation of the disease usually involves symptoms of sinusitis and periorbital cellulitis with facial pain and decreased vision . a nasal endoscopy and a biopsy or a culture of the infected area are essential for the definitive diagnosis . a culture gives us more information about the species of mucorales , but it has a higher rate of false negatives than the biopsy . yohai et al . reported nasal ulceration or necrosis in 48% and 78% respectively in rm . in our case , the initial nasal endoscopy was unremarkable , but we performed a middle meatus culture in view of the high suspicion of rm and the culture revealed a fungus of the order mucorales . the contrast - enhanced sinus ct is helpful to delimit the extension of rm and reveal signs of invasion . magnetic resonance is more sensitive than ct to evaluate intradural and intracranial involvement , the cavernous sinus thrombosis and thrombosis of the cavernous portions of the internal carotid artery . radical resection removing all necrotic tissue should be done and various debridements are usually required . lateral rhinotomy , orbital exenteration and occasionally intracranial surgery are performed depending on the extension of rm . orbital exenteration is necessary when the orbit is infected with limited eye movements in all directions ( frozen eye ) and loss of vision . the surgery that we proposed to our patient was a left paralateral rhinotomy with orbital exenteration and total ethmoidectomy removing all the unviable tissue . amphotericin b is the drug of selection , but it is limited because of its renal and systemic toxic effect . this lipid formulation improves circulation time and concentration in the infected area of the associated amphotericin b. the duration of the treatment is not explicitly established . survival rates in diabetic patients with rm are higher than those in patients with other predisposing factors such as renal failure , hematologic malignancies or autoimmune diseases . a delay in diagnosis and treatment , brain involvement , hemiparesis , bilateral sinus involvement and facial necrosis is also associated with poor survival . rm is a rare and aggressive fungal infection in heart transplant recipients and carries a high mortality rate .

backgroundthe complexity of multiple - item criteria in acute respiratory distress syndrome ( ards ) often causes inconvenience for physicians in the management of patients with severe acute pancreatitis ( sap ) . we evaluated whether serum sp - a levels in the presence of diffuse alveolar damage ( dad ) can be qualitatively assessed for diagnosis of sap - induced ards.material/methodseighty rats were randomly divided into 2 groups ( n=40 each ) the sham - operated ( so ) group and the sap group and then randomly subdivided into 4 subgroups in a time - course manner . furthermore , rats in the sap group were subdivided into the sap induced - ards group ( ards group ) and the sap without ards group ( non - ards group ) according to the diagnostic standard of ards . the diagnostic cut - off values of sp - a for sap - induced ards were determined by the receiver operating characteristic curve ( roc).resultsserum sp - a levels in baseline , so group , sap group , ards group , and non - ards group were 43.1514.29 , 51.9116.99 , 193.435.37 , 198.0 + 29.73 , and 185.743.21 ug / ml , respectively . the best cut - off value for the serum sp - a level for the diagnosis of sap - induced ards was 150 ug / ml and the area under the roc curve of sp - a was 0.88 . the sensitivity , specificity , positive predictive value , negative predictive value , and accuracy of sp - a in the diagnosis of sap - induced ards were 100.0% , 81.8% , 71.4% , 100.0% , and 87.5% , respectively.conclusionsserum sp - a levels may allow the detection of sap - induced ards and may help to support the clinical diagnosis of ards . the optimal serum sp - a cut - off value to discriminate sap - induced ards and other groups ( so group and non - ards group ) is around 150 ug / ml . mucormycosis is an opportunistic acute fungal infection with a high mortality rate seen in immunocompromised patients . it is extremely rare in heart transplant recipients . rhinocerebral mucormycosis ( rm ) is the most frequently observed presentation . we report a case of rm in a heart transplant recipient 5-month after the procedure , with a fatal outcome .

dental erosion is a clinical condition that is currently being studied since researchers have reported an increase in its prevalence . furthermore , dentists are becoming more aware about the etiology and early detection of this condition . acid exposure causes enamel surface demineralization , and long - term demineralization leads to erosive enamel wear as a consequence of progressive mineral loss . dentin erosion presents a different histology lesion , since the progression of demineralization is modulated by the presence of the demineralized organic matrix ( dom ) . the dom may be affected by enzymatic and chemical degradation , which may enhance the progression of dentin wear over time . the primary preventive measure for dental erosion is to reduce the frequency and duration of acid exposure . as it is difficult to control the behavior of the patient , such as the frequency of acid intake or special drinking habits , other strategies have been proposed to control dental erosion that are rather less dependent on the compliance of the patient . the most tested alternative is to increase the acid resistance of the teeth through the application of fluoride . accordingly , highly concentrated fluoride applications such as oral rinses , gels and varnishes have been tested . the application of naf at high concentration is able to promote a caf2-like layer precipitation on enamel . the caf2 globules behave as a physical barrier , inhibiting the contact of the acid with enamel and/or acting as a fluoride reservoir . however , this layer presents low acid resistance , thus , its efficacy in controlling dental erosion is restricted , at least under in vitro conditions . on this basis , recent studies have focused on other fluorides , such as those containing polyvalent metals , which may have a higher efficacy than naf due to surface precipitation or incorporation of ions into sound and demineralized tissue . accordingly , the tif4 has shown a promising erosion - inhibiting effect both in vitro and in situ compared to other fluorides . it is hypothesized that titanium may conflate with phosphate groups , producing an acid - resistant surface coating . most studies have applied fluoride between the erosive challenges , showing a positive effect of different fluoride formulations on the progression of tooth erosion . recently , our research group has shown that an experimental tif4 varnish can be more or at least as effective as a naf varnish in reducing erosive tooth wear when applied only once on a previously sound surface . however , there have been no data published regarding the effect of tif4 and naf varnishes on the reduction of tooth erosion progression when applied on pre - eroded tooth surfaces . patients are usually treated with fluoride to prevent erosion when they have signs of the lesion . therefore , the present study aimed to analyze and compare the effect of tif4 and naf varnishes on enamel and dentin erosion progression in vitro . the null hypothesis tested was that there is no significant difference between tif4 and naf varnishes on the progression of erosive tooth wear when compared to the control . bovine teeth were prepared to obtain 48 enamel and 60 root dentin samples ( 4x4x3 mm ) . the teeth were stored in 0.1% buffered thymol solution ( ph 7.0 ) at 4c . the specimens were cut using an isomet low - speed saw cutting machine ( buehler ltd . , lake bluff , il , usa ) with two diamond discs ( extec corp . , eneld , ct , usa ) , which were separated by a 4 mm thick spacer . the surfaces of the specimens were ground at using water - cooled silicon carbide discs ( 320 , 600 and 1200 grades of al2o3 papers ; buehler , lake bluff , il , usa ) , removing about 200 m of the surface of the tooth . thereafter , the samples were cleaned in an ultrasonic device with deionized water for 5 min . the removal of the cement from the root dentin was verified using a microscope ( x40 magnification ) . two - thirds of the outer surface ( untreated area ) of the specimens were covered with nail varnish in order to create sound areas on both sides of a central band of eroded enamel and dentin . an initial erosive demineralization was created by immersion of the specimens in 0.1% citric acid ( ph 2.5 ) for 30 min at room temperature under gentle agitation , using a shaking bath with horizontal movements ( 60 rpm ) . after acid exposure , enamel and dentin wear ( m ) were quantitatively determined using contact profilometry ( mahr perthometer , mahr ltda , gttingen , germany ) . for the baseline profile measurement ( initial erosive wear ) , the nail varnish was carefully removed using a scalpel and acetone solution ( 1:1 water ) . during the measurement , the dentin specimens were maintained in water ( 100% humidity ) to avoid the dom from shrinking . the diamond stylus was moved from the first reference to the exposed area and then over the other reference area ( 2.5 mm long and 2 mm wide ) . the vertical distance between the horizontal line drawn on the reference areas and the one drawn on the experimental ( eroded ) area was defined as tooth wear using mahr surf xt20 ( software mahr surf xt20 , 2009 , mahr ltda , gttingen , germany ) . the values were averaged ( m ) and used for the random allocation of the samples into the groups ( treatments ) . twelve enamel and 15 dentin specimens were randomly distributed according to the baseline wear of each one of the 4 groups : a 4% tif4 varnish ( 2.45% f- , ph 1.0 ) , a 5.42% naf varnish ( 2.45% f- , ph 5.0 ) , a placebo varnish ( without fluoride ) and no varnish ( control ) . all varnishes contained colophonium , synthetic resin , thickening polymer , essence , artificial sweetener and ethanol . the varnishes were applied in a thin layer using a microbrush , and the specimens were stored in artificial saliva . after 6 h , the varnishes were carefully removed with acetone solution ( 1:1 ) and a scalpel blade , avoiding to touch the enamel surface . the samples were submitted to an erosive demineralization by immersion in 0.1% citric acid ( ph 2.5 ) for 90 s 4 times a day for 5 d. after each demineralization , the specimens were rinsed with deionized water ( 10 s ) and transferred into artificial saliva ( ph 6.8 , 30 ml / specimen , unstirred , 25c ) for 2 h. after the last daily erosive treatment , the specimens were also stored in artificial saliva overnight . the citric acid was renewed at each erosive challenge , and the artificial saliva was replaced daily . the artificial saliva consisted of 0.2 mm glucose , 9.9 mm nacl , 1.5 mm cacl2.2h2o , 3 mm nh4cl , 17 mm kcl , 2 mm nascn , 2.4 mm k2hpo4 , 3.3 mm urea , 2.4 mm nah2po4 , and 11 m ascorbic acid ( ph 6.8 ) . after 5 days of ph cycling , the nail varnish was removed , and 5 profiles were recorded as done at the baseline profile measurement . the values were averaged ( m ) , and the increment value ( final wear baseline wear ) was calculated for enamel and dentin specimens ( with and without the dom in the case of dentin ) and then submitted to statistical analysis . after the profilometric analysis , the dentin samples were submitted to degradation of the collagen fibrils in order to check the influence of the dom on the profile measurement and comparison among the groups . a collagenase enzyme ( type vii from clostridium histolyticum , product no . louis , mo , usa ) , with a collagen digestion activity of 1.98 u/g , solid at 25c , ph 7.5 , in the presence of calcium ions , was used to remove the dom . the collagen layer was removed by adding 100 u / ml of enzyme into artificial saliva containing 20 mm hepes , 0.70 mm cacl2 , 0.20 mm mgcl2.6h2o , 4 mm kh2po4 , 30 mm kcl , 0.30 mm nan3 , and edta - free protease inhibitor cocktail ( complete tm protease inhibitor of cocktail , santa cruz biotechnology , santa cruz , ca , usa ) for 5 d at 37c . the software graphpad instat ( graphpad software , san diego , ca , usa ) was used . the assumptions of equality of variances and normal distribution of data were verified for all the variables tested using bartlett and kolmogorov - smirnov tests , respectively . as the equality of variances was satisfied for enamel , the differences among treatments were analyzed using anova followed by tukey s test . for dentin , the equality of variances was not satisfied , and the differences among treatments were analyzed using kruskal - wallis followed by dunn s test . the sample size was 12 for enamel and 15 for dentin , and the level of significance was set at 5% . bovine teeth were prepared to obtain 48 enamel and 60 root dentin samples ( 4x4x3 mm ) . the teeth were stored in 0.1% buffered thymol solution ( ph 7.0 ) at 4c . the specimens were cut using an isomet low - speed saw cutting machine ( buehler ltd . , lake bluff , il , usa ) with two diamond discs ( extec corp . , eneld , ct , usa ) , which were separated by a 4 mm thick spacer . the surfaces of the specimens were ground at using water - cooled silicon carbide discs ( 320 , 600 and 1200 grades of al2o3 papers ; buehler , lake bluff , il , usa ) , removing about 200 m of the surface of the tooth . thereafter , the samples were cleaned in an ultrasonic device with deionized water for 5 min . the removal of the cement from the root dentin was verified using a microscope ( x40 magnification ) . two - thirds of the outer surface ( untreated area ) of the specimens were covered with nail varnish in order to create sound areas on both sides of a central band of eroded enamel and dentin . an initial erosive demineralization was created by immersion of the specimens in 0.1% citric acid ( ph 2.5 ) for 30 min at room temperature under gentle agitation , using a shaking bath with horizontal movements ( 60 rpm ) . after acid exposure , enamel and dentin wear ( m ) were quantitatively determined using contact profilometry ( mahr perthometer , mahr ltda , gttingen , germany ) . for the baseline profile measurement ( initial erosive wear ) , the nail varnish was carefully removed using a scalpel and acetone solution ( 1:1 water ) . during the measurement , the dentin specimens were maintained in water ( 100% humidity ) to avoid the dom from shrinking . the diamond stylus was moved from the first reference to the exposed area and then over the other reference area ( 2.5 mm long and 2 mm wide ) . five profile measurements were performed at intervals of 0.5 mm . the vertical distance between the horizontal line drawn on the reference areas and the one drawn on the experimental ( eroded ) area was defined as tooth wear using mahr surf xt20 ( software mahr surf xt20 , 2009 , mahr ltda , gttingen , germany ) . the values were averaged ( m ) and used for the random allocation of the samples into the groups ( treatments ) . twelve enamel and 15 dentin specimens were randomly distributed according to the baseline wear of each one of the 4 groups : a 4% tif4 varnish ( 2.45% f- , ph 1.0 ) , a 5.42% naf varnish ( 2.45% f- , ph 5.0 ) , a placebo varnish ( without fluoride ) and no varnish ( control ) . all varnishes contained colophonium , synthetic resin , thickening polymer , essence , artificial sweetener and ethanol . the varnishes were applied in a thin layer using a microbrush , and the specimens were stored in artificial saliva . after 6 h , the varnishes were carefully removed with acetone solution ( 1:1 ) and a scalpel blade , avoiding to touch the enamel surface . the samples were submitted to an erosive demineralization by immersion in 0.1% citric acid ( ph 2.5 ) for 90 s 4 times a day for 5 d. after each demineralization , the specimens were rinsed with deionized water ( 10 s ) and transferred into artificial saliva ( ph 6.8 , 30 ml / specimen , unstirred , 25c ) for 2 h. after the last daily erosive treatment , the specimens were also stored in artificial saliva overnight . the citric acid was renewed at each erosive challenge , and the artificial saliva was replaced daily . the artificial saliva consisted of 0.2 mm glucose , 9.9 mm nacl , 1.5 mm cacl2.2h2o , 3 mm nh4cl , 17 mm kcl , 2 mm nascn , 2.4 mm k2hpo4 , 3.3 mm urea , 2.4 mm nah2po4 , and 11 m ascorbic acid ( ph 6.8 ) . after 5 days of ph cycling , the nail varnish was removed , and 5 profiles were recorded as done at the baseline profile measurement . the values were averaged ( m ) , and the increment value ( final wear baseline wear ) was calculated for enamel and dentin specimens ( with and without the dom in the case of dentin ) and then submitted to statistical analysis . after the profilometric analysis , the dentin samples were submitted to degradation of the collagen fibrils in order to check the influence of the dom on the profile measurement and comparison among the groups . a collagenase enzyme ( type vii from clostridium histolyticum , product no . louis , mo , usa ) , with a collagen digestion activity of 1.98 u/g , solid at 25c , ph 7.5 , in the presence of calcium ions , was used to remove the dom . the collagen layer was removed by adding 100 u / ml of enzyme into artificial saliva containing 20 mm hepes , 0.70 mm cacl2 , 0.20 mm mgcl2.6h2o , 4 mm kh2po4 , 30 mm kcl , 0.30 mm nan3 , and edta - free protease inhibitor cocktail ( complete tm protease inhibitor of cocktail , santa cruz biotechnology , santa cruz , ca , usa ) for 5 d at 37c . the software graphpad instat ( graphpad software , san diego , ca , usa ) was used . the assumptions of equality of variances and normal distribution of data were verified for all the variables tested using bartlett and kolmogorov - smirnov tests , respectively . as the equality of variances was satisfied for enamel , the differences among treatments were analyzed using anova followed by tukey s test . for dentin , the equality of variances was not satisfied , and the differences among treatments were analyzed using kruskal - wallis followed by dunn s test . the sample size was 12 for enamel and 15 for dentin , and the level of significance was set at 5% . the baseline erosive wear means were 12.9 and 11.4 m for enamel and dentin , respectively . the mean increment of enamel wear ( m ) was similar between tif4 and naf varnish . both fluoride varnishes showed significantly less erosion ( 67% and 53% decrease in enamel wear , respectively ) compared to the placebo varnish and the control group ( p<0.0001 ) , which did not differ from each other . table 1meanstandard deviation of the baseline , final and increment enamel erosive wear ( m ) according to each treatment tif nafplacebocontrolbaseline13.31.712.82.712.82.312.52.7final14.72.114.82.016.72.517.02.7increment1.51.1a2.11.7a3.91.1b4.50.9b*distinct lower case letters indicate significant differences among the treatment groups ( anova and tukey s test , p<0.0001 , n=12 ) * distinct lower case letters indicate significant differences among the treatment groups ( anova and tukey s test , p<0.0001 , n=12 ) table 2baseline , final and increment dentin erosive wear means ( m ) , with and without dom ( interquartile range ) , respectively , according to each treatment tif4 nafplacebocontrolbaseline11.311.211.211.2final12.3/13.112.0/13.214.6/16.014.8/15.4increment0.97 ( 0.49 ) 1.87 ( 0.95 ) 1.03 ( 0.68 ) 2.13 ( 0.73 ) 3.53 ( 1.42 ) 4.47 ( 1.91 ) 3.53 ( 1.62 ) 4.36 ( 2.18 ) * distinct lower case letters indicate significant differences among the treatment groups ( kruskal - wallis and dunn , p<0.0001 , n=15 ) * distinct lower case letters indicate significant differences among the treatment groups ( kruskal - wallis and dunn , p<0.0001 , n=15 ) regarding dentin , tif4 and naf presented similar ability to reduce the progression of erosive wear ( 72.5% and 71% decrease in dentin wear , respectively ) , regardless of the presence or absence of the dom , compared to placebo varnish and control group ( p<0.0001 ) , which did not differ from each other . investigations of the potential of tif4 to prevent tooth erosion have been performed since 1997 . it has been speculated that its effect against erosion is due to a high incorporation of fluoride into enamel and a formation of an acid surface precipitation composed by tio2 or hydrated titanium phosphate . our group showed that the tif4 incorporated in an experimental varnish presented a higher protective potential than the tif4 solution on enamel erosion , enamel erosion - abrasion and dentin erosion - abrasion in vitro . we speculate that the better ability of the varnish to adhere to the tooth surface , allowing an increased contact time with the tooth , is responsible for its positive effect . however , our previous studies have focused on the prevention of dental erosion rather than on the control of its progression . the question of the present study was whether tif4 or naf varnishes were also able to reduce the progression of tooth wear when applied on previously eroded surfaces . in our study , we produced a baseline enamel and dentin erosive lesion by aggressive acid exposure done under agitation . fast stirring can increase the five - fold of the enamel wear compared to constant conditions . clinically , this procedure simulates drinking habits such as rinsing , which may increase the risk of erosive wear . the idea behind the procedure was also to simulate a patient with a history of erosive lesions , for whom a fluoride application would be indicated , according to the basic erosive wear examination ( bewe ) guidelines . the varnishes were then applied on the eroded surface only once and removed after 6 h in order to simulate the clinical professional application . in the present study , all fluoridated varnishes had a similar significant potential to reduce enamel and dentin progression , therefore , the null hypothesis can be rejected . however , there was no significant difference between both fluoride varnishes . the results observed for dentin agree with recent data from our group , in which all fluoridated varnishes tested ( experimental tif4 and naf , duraphat and duofluorid varnishes ) were similarly able to significantly reduce dentin wear compared to the placebo varnish , the fluoridated solutions ( tif4 and naf ) and the control group . a limitation of analyzing erosion in dentin is the complexity of the lesion histology , which is characterized by centripetal mineral loss starting in the peritubular areas that progresses to the intertubular area , leading to a bulk tissue loss and the presence of a completely demineralized dentin surface zone that is stable while hydrated . therefore , erosive wear is difficult to quantify since the quality of the remaining organic layer may interfere in the measurement . the limitation of the wear measurement is due to the shrinkage of the dom that may occur under different environments . therefore , to generate reliable data , the profiles must be measured with the samples immersed in water . on the other hand , schlueter , et al . ( 2011 ) advise that the dom should be removed before the profilometric measurement to prevent this problem from occurring . we found that the thickness of the dom of our samples was about 1 m , regardless of the treatment . it is important to highlight that , in the present study , the results were maintained when the wear was measured on dentin without the dom . some studies have shown that the preventive effect of fluorides on dentin erosion is lower compared to enamel and highly dependent on the presence of the organic matrix . dentin erosion is much more complex than enamel erosion because the organic matrix plays an important role in the progression of wear . the dom has a buffering capacity that prevents further demineralization , especially in the presence of fluoride . at least for naf , the enzymatic removal of the organic matrix substantially decreases its effect . considering that it is still unclear if what extent the organic matrix is retained under clinical conditions , it would be interesting to analyze the effect of tif4 varnish applied on eroded dentin with or without dom . in respect to the results of enamel , the present data are not in accordance with a previous study from our group , in which we found significantly less erosion for the enamel samples treated with a tif4 varnish than those treated with a naf ( duraphat ) or naf / caf2 ( duofluorid ) varnish . based on the experimental conditions , we speculate that the glaze - like surface layer produced by tif4 may be thinner and with lower amounts of ti on demineralized enamel surfaces compared to sound ones , as previously shown by chevitarese , et al . , we suggest that the effect of other preventive measures ( professional products ) should be tested on previously eroded tooth surfaces in future studies , since fluoride application is indicated only for patients with a high risk of erosion , according to bewe guidelines . the effect of the tif4 varnish on eroded dentin surfaces in the absence of the dom is another important topic to be further considered . finally , the interaction of this polyvalent metal fluoride in terms of glaze surface layer formation on an eroded tooth should be tested under conditions as close as possible to the clinical situation by simulating general tooth wear ( e.g. erosion and abrasion ) and using in situ models . under the conditions of the present study , it can be concluded that the tif4 varnish was as effective as the naf varnish to reduce the enamel and dentin erosion progression in vitro . the hla - a locus exon 2 region from position 73346 , allele a*01011 , was used for the design of consensus oligos ( figure 1 ) . allele - specific ( variant ) oligos were designed based on single or double nucleotide variants according to alignment to the arbitrarily selected consensus sequence ( hla - a*0201 ) . variant and consensus oligos consisted of 18-mers with a 5 ' amino - modifier having a six - carbon spacer for immobilization ( operon technologies , inc , alameda , ca ) . melting temperatures of oligo probes were maintained as close as possible to a range of 5660c . 350400 pmol/ l oligo probes synthesized in 96-well format were dried and re - suspended in 3 ssc for printing . probes were spotted onto a 3d - link activated slide for covalent immobilization ( motor roller ) using omnigrid robotic printer ( genemachine ) with four printing pins picking up 0.25 l of probe solution and depositing 0.6 nl per spot ( telechem international , inc . ) . spot diameter was 90100 m , spaced at 250 m to prepare 4 16 6 spot / arrays . after printing , slides were kept in a sealed humidifier chamber at room temperature overnight and blocked with 50 mm ethanolamine , 0.1 m tris , ph9 and 0.1% sds at 50c for 15 minutes followed with rinsing in water twice , washing with 4 ssc/0.1% sds 50c for 45 minutes , rinsing with water briefly and centrifuging at 800 rpm for 3 minutes with micro - plate carriers . 12 heterozygous or homozygous samples were tested for oligo nucleotide array hybridization and confirmed by sequence - based typing using the abi prism 3700 - 96 analyzer . in order to generate single strand dna , pcr products from exon 1 to exon 5 of the hla - a locus were amplified with an attachment of t7 promoter to the 5 ' end using 5 ' t7-ex1a-6 primer 5'aaacgacggccagtgaatacgactcactataggcgccagacgccgaggatggcc3 ' ) and three 3 ' primers ( 3 ' ex 5-a 993 - 1 cat tgc tgg cct ggt tct cc ; 3 ' ex 5-a 993 - 2 cat tgc tgg cct ggt tct ctt ; 3 ' ex 5-a 993 - 3 cat tgc tgg cct agt tct ctt ) . pcr reaction was mixed with 25 l of hotstart pcr reagents ( qiagen , ca ) , 5 ng-0.5 g of genomic dna , 5 l of 15 m 5'primer , 5 l of 15 m 3'primer mix and h2o to a 50 l final volume . the reaction was cycled at 95c for 10 minutes , 96c for 35 seconds , 65c for 45 seconds , 72c for 3 minutes , 4 cycle ; 96c for 30 seconds , 60c for 40 seconds , 72c for 3 minutes , 19 cycles and 96c for 30 seconds , 55c for 40 seconds , 72c for 2 minutes , 9 cycles . one l of pcr product from each sample was analyzed using a bioanalyzer on dna7500 chip ( agilent biotechnology ) . the pcr products were then precipitated with etoh at room temperature and re - suspended in depc - treated h2o at 0.1 g/l concentration . in vitro transcription ( ivt ) for each sample , the following reaction mixture was made : 4 l of each 75 mm ntp ( a , g , c and utp ) , 4 l reaction buffer , 4 l enzyme mix ( rnase inhibitor and t7 phage polymerase ) and 1 g purified pcr product in 16 l depc - treated h2o . amplified rna was then purified using trizol reagent according to manufacture instruction ( gibcobrl ) and re - suspended in 40 l of depc water . rna concentrations were estimated by using a bioanalyzer on rna 6000 chip ( agilent biotechnology ) . fluorescence - labeled single strand cdna was generated by reverse transcription ( rt ) and used for hybridization . in the rt reaction , 4 l of first strand buffer , 1 l random hexmer ( 8 g/l ; boehringer mannheim ) , 2 l 10 low t - dntp ( 5 mm a , c and gtp , 2 mm dttp ) , 2 l 1 mm cy - dutp ( cy3 for reference sample or cy5 for test sample unless otherwise specified ) , 2 l 0.1 m dtt , 1 l rnasin and 1.2 g amplified rna in 8 l depc h2o were mixed and heated for five minutes at 65c . this was followed by addition of 1 l superscript ii ( life technology , ) , 40 minutes of incubation at 42c , another 1 ul of superscript ii and 50 more minutes continued incubation at 42c . reactions were stopped by addition of 2.5 l 500 mm edta , 5 l 1 m naoh and heated to 65c for 15 minute to hydrolyze the rna . tris buffer ( 12.5 l of 1 m ) was added immediately to neutralize the ph , and the volume risen to 70 l by adding 35 l of 1 te . the flow through mixed with 200 l 1 te was concentrated to 2040 l using microcon ym-30 column ( millipore ) and further concentrated to 8 ul using speed - vacuum . cy3- and cy5-labeled probes were combined ( 1:1 ratio ) and 5 l 20 ssc , 0.5 l 10% sds and 0.5 l of 4 mg / ml salmon sperm dna were added to the probe for hybridization . prepared probe mixture was applied to an array slide with cover slid and hybridized at 47c for different amounts of time as described in the text . slides were washed with 4 ssc , 2 ssc with 0.1 % sds , 1 ssc , 0.2 ssc and 0.05 ssc sequentially for one minute each step and dried by centrifugation at 800 rpm for 3 minutes . the slides were then scanned for fluorescent signal using a genepix 4000b scanner and the results analyzed using genepix pro3 software ( axon instruments , inc . ) . the hla - a locus exon 2 region from position 73346 , allele a*01011 , was used for the design of consensus oligos ( figure 1 ) . allele - specific ( variant ) oligos were designed based on single or double nucleotide variants according to alignment to the arbitrarily selected consensus sequence ( hla - a*0201 ) . variant and consensus oligos consisted of 18-mers with a 5 ' amino - modifier having a six - carbon spacer for immobilization ( operon technologies , inc , alameda , ca ) . melting temperatures of oligo probes were maintained as close as possible to a range of 5660c . 350400 pmol/ l oligo probes synthesized in 96-well format were dried and re - suspended in 3 ssc for printing . probes were spotted onto a 3d - link activated slide for covalent immobilization ( motor roller ) using omnigrid robotic printer ( genemachine ) with four printing pins picking up 0.25 l of probe solution and depositing 0.6 nl per spot ( telechem international , inc . ) . spot diameter was 90100 m , spaced at 250 m to prepare 4 16 6 spot / arrays . after printing , slides were kept in a sealed humidifier chamber at room temperature overnight and blocked with 50 mm ethanolamine , 0.1 m tris , ph9 and 0.1% sds at 50c for 15 minutes followed with rinsing in water twice , washing with 4 ssc/0.1% sds 50c for 45 minutes , rinsing with water briefly and centrifuging at 800 rpm for 3 minutes with micro - plate carriers . 12 heterozygous or homozygous samples were tested for oligo nucleotide array hybridization and confirmed by sequence - based typing using the abi prism 3700 - 96 analyzer . in order to generate single strand dna , pcr products from exon 1 to exon 5 of the hla - a locus were amplified with an attachment of t7 promoter to the 5 ' end using 5 ' t7-ex1a-6 primer 5'aaacgacggccagtgaatacgactcactataggcgccagacgccgaggatggcc3 ' ) and three 3 ' primers ( 3 ' ex 5-a 993 - 1 cat tgc tgg cct ggt tct cc ; 3 ' ex 5-a 993 - 2 cat tgc tgg cct ggt tct ctt ; 3 ' ex 5-a 993 - 3 cat tgc tgg cct agt tct ctt ) . pcr reaction was mixed with 25 l of hotstart pcr reagents ( qiagen , ca ) , 5 ng-0.5 g of genomic dna , 5 l of 15 m 5'primer , 5 l of 15 m 3'primer mix and h2o to a 50 l final volume . the reaction was cycled at 95c for 10 minutes , 96c for 35 seconds , 65c for 45 seconds , 72c for 3 minutes , 4 cycle ; 96c for 30 seconds , 60c for 40 seconds , 72c for 3 minutes , 19 cycles and 96c for 30 seconds , 55c for 40 seconds , 72c for 2 minutes , 9 cycles . one l of pcr product from each sample was analyzed using a bioanalyzer on dna7500 chip ( agilent biotechnology ) . the pcr products were then precipitated with etoh at room temperature and re - suspended in depc - treated h2o at 0.1 g/l concentration . in vitro transcription ( ivt ) for each sample , the following reaction mixture was made : 4 l of each 75 mm ntp ( a , g , c and utp ) , 4 l reaction buffer , 4 l enzyme mix ( rnase inhibitor and t7 phage polymerase ) and 1 g purified pcr product in 16 l depc - treated h2o . amplified rna was then purified using trizol reagent according to manufacture instruction ( gibcobrl ) and re - suspended in 40 l of depc water . rna concentrations were estimated by using a bioanalyzer on rna 6000 chip ( agilent biotechnology ) . fluorescence - labeled single strand cdna was generated by reverse transcription ( rt ) and used for hybridization . in the rt reaction , 4 l of first strand buffer , 1 l random hexmer ( 8 g/l ; boehringer mannheim ) , 2 l 10 low t - dntp ( 5 mm a , c and gtp , 2 mm dttp ) , 2 l 1 mm cy - dutp ( cy3 for reference sample or cy5 for test sample unless otherwise specified ) , 2 l 0.1 m dtt , 1 l rnasin and 1.2 g amplified rna in 8 l depc h2o were mixed and heated for five minutes at 65c . this was followed by addition of 1 l superscript ii ( life technology , ) , 40 minutes of incubation at 42c , another 1 ul of superscript ii and 50 more minutes continued incubation at 42c . reactions were stopped by addition of 2.5 l 500 mm edta , 5 l 1 m naoh and heated to 65c for 15 minute to hydrolyze the rna . tris buffer ( 12.5 l of 1 m ) was added immediately to neutralize the ph , and the volume risen to 70 l by adding 35 l of 1 te . the flow through mixed with 200 l 1 te was concentrated to 2040 l using microcon ym-30 column ( millipore ) and further concentrated to 8 ul using speed - vacuum . cy3- and cy5-labeled probes were combined ( 1:1 ratio ) and 5 l 20 ssc , 0.5 l 10% sds and 0.5 l of 4 mg / ml salmon sperm dna were added to the probe for hybridization . prepared probe mixture was applied to an array slide with cover slid and hybridized at 47c for different amounts of time as described in the text . slides were washed with 4 ssc , 2 ssc with 0.1 % sds , 1 ssc , 0.2 ssc and 0.05 ssc sequentially for one minute each step and dried by centrifugation at 800 rpm for 3 minutes . the slides were then scanned for fluorescent signal using a genepix 4000b scanner and the results analyzed using genepix pro3 software ( axon instruments , inc . ) .

objective this study assessed the effect of fluoride varnishes on the progression of tooth erosion in vitro . material and methods : forty - eight enamel and 60 root dentin samples were previously demineralized ( 0.1% citric acid , ph 2.5 , 30 min ) , leading to a baseline and erosive wear of 12.9 and 11.4 m , respectively . the samples were randomly treated ( 6 h ) with a 4% tif4 varnish ( 2.45%f- , ph 1.0 ) , a 5.42% naf varnish ( 2.45%f- , ph 5.0 ) , a placebo varnish and no varnish ( control ) . the samples were then subjected to erosive ph cycles ( 4x90 s / day in 0.1% citric acid , intercalated with artificial saliva ) for 5 days . the increment of the erosive tooth wear was calculated . in the case of dentin , this final measurement was done with and without the demineralized organic matrix ( dom ) . enamel and dentin data were analyzed using anova / tukey s and kruskal - wallis / dunn tests , respectively ( p<0.05).results the tif4 ( means.d : 1.51.1 m ) and naf ( 2.11.7 m ) varnishes significantly reduced enamel wear progression compared to the placebo varnish ( 3.91.1 m ) and control ( 4.50.9 m ) . the same differences were found for dentin in the presence and absence of the dom , respectively : tif4 ( average : 0.97/1.87 m ) , naf ( 1.03/2.13 m ) , placebo varnish ( 3.53/4.47 m ) and control ( 3.53/4.36 m).conclusion the tif4 and naf varnishes were equally effective in reducing the progression of tooth erosion in vitro . detection of unknown single nucleotide polymorphism ( snp ) relies on large scale sequencing expeditions of genomic fragments or complex high - throughput chip technology . we describe a simplified strategy for fluorimetric detection of known and unknown snp by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes . the array consists of two sets of 18-mer oligonucleotide probes . one set includes overlapping oligos with 4-nucleotide tiling representing an arbitrarily selected " consensus " sequence ( consensus - oligos ) , the other includes oligos specific for known snp within the same genomic region ( variant - oligos ) . fluorescence - labeled dna amplified from a homozygous source identical to the consensus represents the reference target and is co - hybridized with a differentially - labeled test sample . lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant - oligos designates a known allele . lack of hybridization to consensus- and variant - oligos indicates a new allele . detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal . this method can identify unknown snp in heterozygous conditions with a sensitivity of 82% and specificity of 90% . this strategy should dramatically increase the efficiency of snp detection throughout the human genome and will decrease the cost and complexity of applying genomic wide analysis in the context of clinical trials .

cancer is one of the most common malignancies worldwide ; it is the leading cause of death in economically developed countries and the second leading cause of death in developing countries . approximately 12.7 million new cases of cancer and 7.6 million cancer - related deaths were reported in 2008 . despite the efforts exerted by many researchers to elucidate the mechanism of carcinogenesis , environmental factors , diet , lifestyle , and smoking and drinking habits have been implicated in the development of cancer [ 3 , 4 ] . various epidemiological studies have revealed that inflammation - associated factors , such as interleukin- ( il- ) 1 , il-6 , il-10 , and tumor necrosis factor- , are associated with cancer tumorigenesis . interleukin-17 ( il-17 ) is a proinflammatory cytokine that serves important functions in inflammation , autoimmune disorders , and cancer . the il-17 cytokine family consists of six members ( il-17a to il-17f ) and five receptors ( il-17ra to il-17rd and sef ) [ 7 , 8 ] . these cytokines are primarily produced from a subset of cd4 + effector cells known as th17 cells [ 9 , 10 ] . clinical studies have shown increased il-17 expression in malignant tumors [ 1114 ] . single nucleotide polymorphisms ( snps ) can alter gene functions and protein expression , which influence cell proliferation and increase cancer risk . the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms are the most common loci associated with il-17 activity and cancer risk . in 2009 , shibata et al . conducted the first study and reported a positive relationship between gastric cancer and the il-17a rs2275913g > a polymorphism in a japanese population . but no significant association was found between gastric cancer and polymorphisms of il-17f rs763780 t > c . many epidemiological studies have focused on the association of the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms with cancer risk therefore , we performed a meta - analysis to clarify the possible association of the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms with cancer risk . the pubmed , embase , and chinese national knowledge infrastructure databases were searched using the terms cancer , tumor , the related articles option was also used in each research article to find potential relevant studies on the same topic . only studies published in english or chinese the inclusion criteria in this meta - analysis were as follows : ( a ) researches that focused on population , ( b ) studies that evaluated the association of the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms with cancer risk , ( c ) case - controls studies , and ( d ) studies that contain available genotype frequency to estimate the odds ratio ( or ) and 95% confidence intervals ( cis ) . the largest or the most recent publication was selected when some data were the same or overlapped . two investigators ( yu - ming niu and hua yuan ) independently extracted the following information from each eligible publication : first author 's name , publication year , country of origin , ethnicity of the individuals involved ( categorized as asian or caucasian ) , source of controls , number of cases and controls , number of genotypes cases and controls , hardy - weinberg equilibrium ( hwe ) and minor allele frequency ( maf ) , and cancer category . the strength of the association of the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms with cancer risk was assessed by calculating crude ors with 95% cis . heterogeneity was calculated based on the i statistic with low , moderate , and high i values of 25% , 50% , and 75% , respectively [ 16 , 17 ] . when i 50% ( which indicated a lack of heterogeneity ) , the or estimation of each model was calculated by using the fixed - effects model ( mantel - haenszel method ) ; otherwise , the random - effects model ( dersimonian and laird method ) was used . sensitivity analyses were assessed by deleting each study to reflect the influence of individual datasets on the pooled ors . statistical analysis was performed using stata version 11.0 ( stata corporation , college station , tx , usa ) with two - sided p values . a total of 185 relevant studies were found with the research words and manual research . after careful review , 10 published case - control studies involving 4,516 cases and 5,645 controls met our inclusion criteria [ 15 , 2028 ] . we found 10 and 7 eligible studies with adequate genotype and research subjects according to il-17a rs2275913g > a and il-17frs763780t > c polymorphism . nine studies involved asian populations ( seven involved the chinese population ) , and one study involved a caucasian population . diverse genotyping methods were used , including polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) [ 23 , 27 ] , taqman [ 21 , 24 , 28 ] , polymerase chain reaction - sequence specific primers ( pcr - sscp ) [ 15 , 25 ] , massarray [ 20 , 22 ] , and snapshot snp assay methods in eligible publications . the genotypic distribution of controls in only two and three studies deviated from hwe in the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms , respectively . the il-17a rs2275913g > a polymorphism showed significant associations with cancer risk in all populations ( for a versus g : or = 1.28 , 95% ci : 1.161.41 , p < 0.001 , i = 61.1% ; for ga versus gg : or = 1.12 , 95% ci : 1.021.23 , p = 0.015 , i = 27.8% ; for aa versus gg : or = 1.71 , 95% ci : 1.382.41 , p < 0.001 , i = 69.6% ; for ga + aa versus gg : or = 1.23 , 95% ci : 1.131.34 , p < 0.001 , i = 6.4% ( figure 2 ) ; and for aa versus gg + ga : or = 1.62 , 95% ci : 1.272.07 , p < 0.001 , i = 81.4% ) . the succeeding stratified analysis according to hwe , ethnicity , and study design subgroup also presented that the il-17a rs2275913g > a polymorphism may be a strong risk factor in the development of cancer , especially gastric cancer , in the chinese population . statistical analysis also indicated that the il-17f rs763780t > c polymorphisms were significantly associated with cancer risk , particularly gastric cancer ( for c versus t : or = 1.29 , 95% ci : 1.141.46 , p < 0.001 , i = 0% ; for tc versus tt : or = 1.33 , 95% ci : 1.131.55 , p < 0.001 , i = 21.6% ; for cc versus tt : or = 1.40 , 95% ci : 1.041.88 , p = 0.026 , i = 0% ; for tc + cc versus tt : or = 1.34 , 95% ci : 1.161.55 , p < 0.001 , i = 16% ( figure 3 ) ) . a single study involved in the meta - analysis was deleted each time to reflect the influence of the individual data set on the pooled ors , and the corresponding pooled ors were not qualitatively altered . this indicated that the results about the association between il-17 gene polymorphisms and cancer risk were statistically robust ( figures 4 and 5 ) . funnel plot and egger 's test were performed to estimate the publication bias of literature . publication bias was detected in the meta - analyses on the allele contrast , homozygote ( aa versus gg ) , dominant , and recessive models of the il-17a rs2275913g > a polymorphism ( figure 6 for ga + aa versus gg model ) , except for the ga versus gg model ( p = 0.652 ) . stratified analyses were conducted only with the hwe and chinese population , but the results were not substantially different . for the il-17f rs763780t > c polymorphism , the funnel plots did not show any asymmetrical evidence in all genetic models ( figure 7 ) . the result was further supported by analysis using egger 's tests ( p = 0.102 for c versus t ; p = 0.185 for tc versus tt ; p = 0.382 for cc versus tt ; p = 0.114 for tc + cc versus tt ( figure 7 ) ; p = 0.792 for cc versus tt + tc ) . carcinogenesis is a multistep process that involves numerous factors , such as smoking , drinking , xenobiotics infections , nutrition deficiency , and host genetic factor . cancer - related inflammation factors have been recently confirmed to increase the risk of developing malignant tumors . il-17 is a relatively novel cytokine family that is connected with adaptive and innate immune systems . il-17a and il-17f are members of the il-17 cytokine family that are responsible for the pathogenic activity of il-17 cells , the lineage of cd4 effector cells , and multiple proinflammatory mediators . genetic polymorphisms of the il-17a and il-17f cytokines could change the function and expression of cytokines , which influence the activity of ils [ 12 , 30 , 31 ] . several studies have revealed that il-17a and il-17f polymorphisms are associated with gastric cancer , breast cancer , and so on . however , the results of these studies are inconsistent . in 2009 , shibata et al . were the first to report that the aa homozygote was significantly correlated with the development of gastric cancer compared with the common homozygous genotype ( gg ) in a japanese population ( or = 3.02 , 95% ci : 1.864.91 ) . one year later , chen et al . also found a positive relationship between gastric cancer and the il-17a rs2275913g > a polymorphism in a chinese han population with a drinking habit ( for a versus g : or = 1.37 , 95% ci : 1.071.76 ) . , rafiei et al . , zhang et al . , and zhuet al . in gastric cancer . furthermore , the mutation of il-17a rs2275913g > a locus was also demonstrated as tumorigenic for bladder cancer , breast cancer , and cervical cancer . however , another article detected no significant association between the il-17a rs2275913g > a polymorphism and gastric cancer risk . regarding the il-17f rs763780t > c polymorphism , wu et al . found that the ct and cc genotypes are associated with an increased risk of gastric cancer compared with the tt genotype ( or = 1.51 , 95% ci : 1.221.87 for ct ; or = 1.61 , 95% ci : 1.032.51 for cc ) . by contrast , zhou et al . showed that bladder cancer patients have significantly higher frequencies of t allele than controls . this result indicates that this t allele is significantly associated with bladder cancer ( or = 1.46 , 95% ci : 1.072.00 ) . furthermore , other researches did not find any significant association between the il-17f rs763780t > c polymorphism and cancer risk [ 15 , 20 , 22 , 24 , 26 ] . to the best of our knowledge , this meta - analysis is the first to determine the association of il-17 polymorphisms with cancer risk . this study focused on two common il-17 polymorphisms , namely , il-17a rs2275913g > a ( 10 studies with 4,516 cases and 5,645 controls ) and il-17f rs763780t > c ( 7 studies with 2,863 cases and 3,773 controls ) . significant associations were found between the il-17a rs2275913g > a polymorphism and cancer risk in all five genotype models of total populations . besides , we also detected some association between the il-17f rs763780t > c polymorphism and the risk of asians ( chinese ) , population - based and hospital - based controls , and gastric cancer or other cancers in the subgroup analysis by hwe publications , ethnicity , control design , and cancer category . interestingly , nine researches focused on asian population ; the results of our meta - analysis demonstrated that the il-17a rs2275913g > a polymorphism may be a stranger canner risk for asian ethnicity ( including chinese ) . moreover , the category of control design did not influence the results ; not only the population - based but also the hospital - based controls all showed that significant association existed between il-17a rs2275913g > a polymorphism and cancer risk . in all selected publications , seven studies focused on gastric cancer and the results also indicated that il-17a rs2275913g > a polymorphism plays an important role during the development of gastric cancer . for il-17f rs763780t > c polymorphism , all selected studies were conducted in asian population and significant association only was found in codominant model ( cc versus tt ) in total population . furthermore , subgroup analyses revealed a significantly increased risk of gastric cancer with il-17f rs763780t > c polymorphism in four models . a combination of relative genotypes may be a higher risk factor than a single locus genotype . linkage disequilibrium and haplotype analyses of the two polymorphisms were not conducted because of the lack of original data on the individual genotypes from the included studies . third , these results were based on unadjusted estimates , and the evaluations were limited without the effects of gene - gene and gene - environment interactions . finally , most of the included studies had been conducted on asians but not on caucasians and africans , and the association between ethnicity variation and cancer risk could not be explored deeply . in conclusion , despite these limitations , the present meta - analysis demonstrates that the il-17a rs2275913g > a polymorphism is associated with cancer development . furthermore , the il-17f rs763780t > c polymorphism may be a potential risk factor in the development of gastric cancer . in the future , large - scale , case - control , and well - designed studies must be conducted to validate the findings of our meta - analysis and to comprehensively understand the potential gene - gene and gene - environment interactions between il-17 polymorphisms and cancer risk . vibrissae provide input to specific thalamic nuclei and these nuclei have well - defined connections with the somatosensory cortical region where sensory information is processed ( woolsey and van der loos , 1970 ) . these connections have a somatotopic organization , and sensory information from each vibrissa reaches a specific barreloid in the thalamic ventrobasal ( vb ) complex and is relayed to a specific cortical barrel field in the primary somatosensory cortex . during their developmental outgrowth , thalamocortical axons are guided by a variety of cues ( molnr et al . , 2012 ) . after crossing the subpallial - pallial border , thalamocortical axons advance within the intermediate zone ( iz ) and , approaching the cortex , they accumulate below the cortical plate ( cp ) at the subplate ( ghosh et al . , the interaction of thalamocortical axons with the subplate is one of the most enigmatic processes in the development of thalamocortical fibers . even in reeler or p35 knockout ( ko ) mice , where the subplate is aberrantly located , thalamocortical axons cross the cp toward the misplaced subplate before connecting to their final targets ( hoerder - suabedissen and molnr , 2015 ) . however , the molecular mechanisms that control thalamocortical axon - subplate interaction and ultimately the correct targeting of thalamic projections are largely unknown . in the developing brain , bioactive phospholipids like lysophosphatidic acid ( lpa ) play important roles in cortical migration ( fukushima et al . , 2000 ) and neuronal apoptosis ( kingsbury et al . , 2003 ) . these effects are mediated by lpa receptors , namely lpa1-r and lpa2-r , which are expressed in the developing brain ( kingsbury et al . , 2003 ) . lpa is a well - described repellent factor for axons , eventually leading to growth cone ( gc ) collapse ( campbell and holt , 2003 ) . however , while in vitro experiments suggested an involvement of lpa1-r in lpa - mediated axonal retraction , deletion of specific lpa receptors did not lead to significant alterations of fiber tracts in the brain ( contos et al . , 2002 ) and did not affect inhibitory lpa effects on retinal gcs ( birgbauer and chun , 2010 ) . lpa is enzymatically inactivated by dephosphorylation via lipid phosphate phosphatases ( lpps ) , which are cell - surface lipid enzymes and constitute a large family of phosphatidic acid phosphatases ( pap2 ) . recently , it was shown that lpa signaling at the synapse is controlled by prg-1/lppr4 ( trimbuch et al . , 2009 ) , which belongs to a new class of lpa - interacting molecules ( plasticity - related genes , prgs ) sharing transmembrane features of the lpps ( bruer et al . , 2003 ) but which seem to have a different mode of action ( mcdermott et al . , 2004 ) . among these , prg-2/lppr3 is expressed in developing thalamocortical axons and has a high expression level in gcs . using both prg-2 animals and a thalamus - specific prg-2/gbx2/r26 mouse line , we set out to analyze the role of this new lpa regulatory mechanism in outgrowing axons . prg-2/lppr3 is prominently expressed in the thalamus ( as shown by in situ hybridization by http://www.genepaint.org ; figure s1b , available online ) and in developing thalamocortical fibers ( figures 1a and s1a ; for antibody specificity see figures s1g s1i ) , where prg-2 co - localized with the thalamic axonal marker l1 ( robichaux et al . , 2014 ) . according to this expression pattern , we hypothesized that prg-2 plays a role in the developing thalamocortical projection . we therefore performed biocytin tracing studies in thalamocortical brain slices of wild - type ( wt ) and prg-2 mice at embryonic day ( e)17 ( figures 1d , 1e , 1h , and 1i ) , a time point when thalamocortical axons accumulate below the subplate ( molnr et al . , 1998 ) . slices were injected with alexa 488-labeled biocytin into the vb complex of the thalamus and were incubated for 6 hr to allow for anterograde biocytin transport . this procedure combines the advantages of maintaining slice integrity with precise tracer application in the vb . while wt thalamocortical fibers were restricted to the iz ( figures 1d and 1h ) , as described by others ( molnr et al . , 1998 ) , prg-2 fibers aberrantly protruded into the cp ( figures 1e and 1i ) . to further substantiate this finding , we used a conditional prg-2 mouse line ( figures s1e and s1f ) , which was cross - bred with a gbx2 line , resulting in thalamus - specific creer expression ( chen et al . 2013 ) , and an r26 line ( madisen et al . , 2010 ) , which produces red fluorescent protein ( rfp ) upon cre - mediated recombination . using thalamus - specific prg-2-deficient mice ( prg-2/gbx2/r26 ) , we analyzed the effects of thalamus - specific prg-2 deletion induced by tamoxifen treatment at e12.5 ( prg-2 ; see also figures s1c and s1d ) , as described ( normand et al . , 2013 ) . here , we found a similar aberrant phenotype as observed in constitutive prg-2 mice ( figures 1f and 1j ) . to prove that not only a fiber subpopulation aberrantly protruded into the cp , we analyzed the full thalamocortical projection , which was displayed by the rfp reporter , and confirmed the aberrant phenotype ( figure 1 g ) . quantitative assessment of these slices revealed a significant disruption of the thalamocortical projection in constitutive prg-2 mice as well as in prg-2 mice ( figure 1k ) . to demonstrate that misrouting was also present in vivo , we applied neurovue tracer in the thalamic vb at e17 in fixed whole brains and analyzed thalamocortical axons . while thalamocortical fibers formed well - defined projections restricted to the iz in wt animals ( figure 1l ) , in prg-2 mice , thalamocortical fibers invaded the cp , showing a similar misrouting as observed in biocytin - traced thalamocortical slices ( figure 1 m ) . to avoid remote effects , e.g. , by prg-2 deletion in the cortex , we analyzed the thalamocortical projection in vivo in prg-2 mice , where the rfp reporter delineated the thalamocortical fibers with high accuracy , as described by others ( normand et al . , 2013 ) . here , prg-2-deficient thalamocortical fibers aberrantly invaded the cp , while prg-2-expressing fibers were restricted to the iz ( figures 1b and 1c ) . interestingly , in prg-2 animals , the subplate had a normal appearance ( figures s1j and s1k ) and the cortical organization , as well as the synaptic connectivity , was not altered ( figures s1m s1 t ) , ruling out possible subplate or cortical defects that could induce mistargeting of thalamocortical fibers ( ghosh et al . , since thalamocortical projection defects observed at prenatal stages might be a temporary feature that eventually disappears after formation of thalamocortical connections , we analyzed the thalamocortical fiber tract at postnatal day ( p)5 , a time point at which the connections of these fibers are established ( molnr et al . we applied lipophilic tracers in different brain areas ( motor cortex , somatosensory cortex , and visual cortex ) at p5 and found that retrogradely labeled thalamocortical fibers derived from their corresponding thalamic nuclei grossly maintained their termination pattern ( figures s2a s2c ) . to analyze whether the thalamocortical projection to the somatosensory cortex follows the well - established somatotopic termination pattern to the appropriate barrels , we inserted lipophilic tracers in the vb and visualized the thalamic projection in the corresponding barrels in tangential cut cortical slices . as shown in figure 1n , in the somatosensory cortex of wt animals lipophilic tracer - stained axons were restricted to their corresponding barrels . however , in prg-2 mice , thalamic axons readily crossed barrel borders aberrantly invading neighboring barrels ( figures 1o , 1p , and s1l ) . to further assess misrouting in prg-2 mice , we performed retrograde tracing of thalamocortical fibers by paired injection of retrogradely transported fluorescent latex microbeads into adjacent cortical barrels with 400 m separation between injection sites . ( 1995 ) , even injections placed in close proximity ( 75120 m apart ) resulted in segregated neuronal clusters of retrogradely labeled cells in the thalamic vb ( agmon et al . , 1995 ) . while in wt animals , even nearby injections of differently labeled fluorescent beads ( red and green fluorescence ; see figure s2d ) resulted in labeling of distinct subpopulations in the corresponding vb thalamic nuclei ( figures 1q and 1s ) , in prg-2 mice , injections even at distant sites ( see figure s2e ) resulted in double labeling of the corresponding subpopulations ( figures 1r and 1 t ; exemplarily retrograde stained neurons in the thalamus are shown in figures s2f and s2 g ) . quantitative assessment of double - labeled neurons revealed a significantly higher number in prg-2 animals , while the total number of retrogradely traced neurons did not differ between genotypes ( figures 1u and 1v ) . thus , prg2 depletion in mice leads to a misrouting in thalamocortical projections . to test whether the aberrant thalamic projection in prg-2 mice results from absence of prg-2 in thalamocortical axons , we used thalamocortical slice cultures of e15 wt and prg-2 mice at the peak of thalamocortical outgrowth . the thalamocortical projection was visualized by co - electroporation of a gfp - expressing construct into the thalamic vb at e15.5 . after 48 hr , slice cultures were fixed and the axonal projection was analyzed ( figure 2a ) . in wt slices , thalamocortical fibers were restricted to the iz ( figure 2b ) , while prg-2 thalamocortical axons aberrantly invaded the cp ( figure 2c ) . however , when prg-2 was re - expressed ( together with gfp ) in thalamocortical axons , these fibers were again restricted to the iz and did not show obvious differences to wt fibers ( figures 2d and s3a ) . quantitative analysis of thalamocortical cultures confirmed the significant amount of aberrant thalamocortical projections and the rescued targeting of these axons after prg-2 re - expression ( figure 2e ) . in sum , re - expression of prg-2 in thalamocortical axons was sufficient to completely abrogate aberrant targeting of the thalamocortical fiber projection in slices from prg-2 mice . since prg-2 is a member of the family of lpa - interacting molecules ( trimbuch et al . , 2009 ) , we analyzed the presence of autotaxin ( atx , enpp-2 , lysopld ) , the lpa - synthesizing enzyme , in the somatosensory cortex at e16 . atx is expressed by subplate neurons , as has been shown on the mrna level ( hoerder - suabedissen et al . , 2013 ) , and by a gfp reporter at corresponding age ( figures s4a and s4b ) . due to its secreted and diffusible nature , we found strong atx expression at the lower border of the cp and at the upper border of the iz ( figure 3a ) . here , atx immunoreactivity overlaps with that of lpa and prg-2 , which was expressed in fasciculated thalamocortical fibers in the iz ( figures 3b3d ; higher magnification in figures 3e3h ) . these findings are in line with a putative role of atx / lpa in restricting prg-2-expressing thalamocortical fibers to the iz . moreover , lpa - degrading enzymes like lpp1 and lpp3 are strongly expressed in the iz ( figures s6n and s6o ) , which suggests that proper regulation of phospholipids allows thalamocortical axons to occupy a corridor in the iz , possibly contributing to the fasciculated appearance of the thalamocortical projection . we therefore perturbed phospholipid signaling and analyzed the effect of inhibited lpa synthesis on the correct targeting of thalamocortical axons . we applied a recently characterized atx blocker ( pf8380 ) ( gierse et al . , 2010 ) , which was shown to effectively decrease lpa concentrations ( vogt et al . , 2015 ) , after atx inhibition ( 0.1 m ) , wt thalamocortical fibers aberrantly invaded the cp ( figure 3j ) , showing a similar phenotype as observed in slices from prg-2 mice ( figures 1e and 1i ) . quantitative analysis revealed that this phenotype was observed in more than 92% of all pf8380-treated slices ( figure 3l ) . to further study whether this effect was due to cortical lpa production inhibition of atx in the cp was sufficient to induce an aberrant targeting of thalamocortical fibers ( figure 3k ) . in contrast , pf8380 injection into the thalamus did not significantly alter the thalamocortical targeting , supporting a role for cortical - derived lpa in thalamocortical guidance ( figure 3l ) . taken together , our data provide strong evidence that correct guidance of thalamic fibers to the cortex depends on subplate - derived lpa , produced by atx , and on the presence of prg-2 on thalamic fibers . the role of lpa in gc collapse is well described ( campbell and holt , 2003 ) ; however , the molecular mechanism transducing lpa action from the membrane to the cytoskeleton is not well understood . while specific , g protein - coupled lpa receptors were reported not to affect lpa - induced collapse of retinal gcs ( birgbauer and chun , 2010 ) , authors of the same group showed that downstream inhibition of the g12/13-rho - rock pathway was capable of inhibiting lpa - induced retinal gc collapse ( fincher et al . , 2014 ) . this is in line with reports of downstream lpa signal transduction acting on the cytoskeleton ( campbell and holt , 2003 ) . to analyze the role of axonal prg-2 in lpa - mediated thalamic axon guidance , we embedded thalamic explants in matrigel with a polarized concentration of lpa ( distance to tf - lpa - containing matrigel was 401.6 53.63 m for wt explants and 475 146.80 m for prg-2 explants ) and on one side , the matrigel contained 10 m fluorescent - labeled lpa ( tf - lpa ) , while the adjacent side did not contain lpa ( see figures 4a s5e , and 4d2 for the border of the tf - lpa - containing matrigel region at higher magnification ) . the 3d matrigel structure , where fibers are able to radially expand , allows analysis of fiber growth into the lpa - rich zone and the adjacent control zones . however , while wt axons were not able to invade this lpa - rich zone , prg-2 axons grew far inside it ( figures 4c and 4d1 ) . quantitative analysis of fibers entering the lpa - rich zone at different distances revealed significantly higher invasion by fibers from prg-2 thalamic slices ( figure 4e ) , while no difference between wt and prg-2 axon fiber outgrowth was observed in the adjacent , non - lpa - containing control side ( figure 4f ) . direct comparison of lpa - containing and control sides revealed that wt axons were significantly repelled by the lpa - rich zone , while prg-2 axons were not affected ( figure 4 g ) . since prg-2 deficiency allowed axons to enter the lpa - rich zone , which is repulsive for wt axons , these data suggest that prg-2 is critically involved in mediating the repulsive lpa sensitivity in thalamic axons . during normal development , thalamocortical axons do not collapse at the border to the cp but rather turn away from this border and continue to grow in the iz until reaching their final target . this indicates that lpa provided by the subplate induces the turning of axon gcs when thalamocortical fibers approach lpa - rich areas . to mimic this situation in our in vitro outgrowth assay , we exposed thalamocortical explants to lower lpa concentrations ( 1 m lpa ; figure 4h ) . here , wt axons displayed a typical turning behavior in front of the lpa - rich zone ( figures 4i and 4k ) . to confirm this observation axon gcs were exposed to a low , but continuous , lpa concentration ( 500 nm lpa ) . to avoid bias by perfusion flow , which may repel axons , gcs were exposed to an lpa - covered pipette tip at 40 m distance ( see figure s5f for experimental overview ) . while wt axons stopped growing and turned away from the lpa source , this behavior was not observed for prg-2 axons , which even seemed to be attracted by lpa ( figures 4l4n ; movies s1 and s2 ) . quantitative analysis revealed a significant repulsion of wt axons by 500 nm lpa , which was not observed in prg-2 axons ( figure 4o ) . however , prg-2 axons were still repelled by other guidance cues like sema3a ( figures s5 g and s5h ) , which are well - described repulsive cues ( mintz et al . , 2008 ) . in sum , these data indicate that prg-2 at the gc mediates the repellent effect of lpa for axonal guidance . prg-2/lppr3 has an intracellular 400 aa - long c - terminal tail , which is putatively involved in signal transduction . to determine interaction partners of this intracellular domain , we first performed a y2h screen using this prg-2 c - terminal tail as a bait and found a positive interaction with the ferm domain of radixin ( rdx ) ( fehon et al . , 2010 ) . using immunoprecipitation ( ip ) on e17 brain lysate this interaction is specific for prg-2 since only prg-1 and prg-2 possess an intracellular c terminus , and prg-1 is not expressed at embryonic ages ( figure s6j ) . to further elucidate the role of this interaction in proper guidance of thalamocortical fibers to the developing cortex , we performed co - localization studies at e16 and found co - expression of prg-2 and rdx in the thalamocortical tract ( figures 5b , 5c , s6k , and s6i ) . expression analysis of prg-2 and rdx showed a similar expression pattern , where expression for both molecules increased from e11.5 , a time point where thalamic neurons are already present and start to differentiate ( figure s6f ) . to prove that the rdx / prg-2 interaction in fact occurs in the axon gc , we performed subcellular immunofluorescent co - localization studies of rdx and prg-2 and found a strong signal in gcs ( figure 5d ) . here , prg-2 is expressed at the gc membrane and co - localizes with rdx , which shows an asymmetric distribution , as described by others ( mintz et al . , 2008 ) . rdx belongs to the ezrin , radixin , and moesin ( erm ) protein family . however , due to their embryonal expression pattern at e14.5 , ezrin and moesin do not seem to be involved in the outgrowth of the thalamocortical projection ( figures s6l and s6 m ) . the c terminus of rdx can directly bind to filamentous actin ( f - actin ) , thereby acting as a crosslinker between the cortical f - actin cytoskeleton and the plasma membrane ( fehon et al . , the activation of rdx involves the phosphorylation of a critical c - terminal threonine residue ( t564 ) , which is highly conserved in the erm protein family . therefore , we analyzed the role of prg-2 in rdx activation using a specific antibody that recognizes the phosphorylation of t564 in rdx , t567 in ezrin , and t558 in moesin ( hausrat et al . , 2015 ) . interestingly , we found significantly lower phosphorylated erm ( perm ) levels in prg-2 neurons ( figures 5e and 5f ) , indicating diminished activation of erm proteins . to prove the role of lpa in erm activation , we assessed the effect of different lpa concentrations . while 100 nm lpa already revealed an increase in erm phosphorylation , 1 m lpa induced a significant and robust increase in perm levels ( figures 5 g and 5h ) . we further analyzed perm levels over time and found a fast and constant increase in erm phosphorylation in the course of lpa stimulation ( figures 5i , 5j , and s6a s6c ) . to understand the functional significance of the lpa - dependent perm expression , we analyzed the rdx / prg-2 interaction under lpa - free conditions and after 1 m lpa stimulation for 15 min . here , we used rdx - gfp transfected , stable prg-2-expressing hek cells , which have comparable prg-2 expression , and performed coips of rdx and prg-2 using an anti prg-2 antibody ( figure 5k ) . quantitative analysis ( rdx levels were adjusted according to the prg-2 ip signal ) revealed a significantly increased rdx / prg-2 interaction upon 1 m lpa stimulation ( figure 5l ) . interestingly , analysis of the ip product revealed that phosphorylation of the prg-2-interacting rdx was significantly higher upon lpa stimulation when compared to serum - starved cells ( figures 5 m and 5n ) . since we could show that rdx binds to the intracellular c terminus of prg-2 ( figure s6d ) , to further analyze whether rdx phosphorylation is a prerequisite for prg-2 binding and to determine the prg-2 binding sites , we performed a systematic peptide scan using a celluspot array format ( spot ) and mapped the rdx / prg-2 interaction ( figure 5o ) . here , prg-2 intracellular c - terminal overlapping peptides ( 69 overlapping peptide fragments , 14 aa in length ) were analyzed regarding their binding affinity to rdx - gst or to rdx - gst , its non - phosphorylated form . we detected six putative interaction sites that showed significant difference in their binding to rdx - gst or to rdx - gst , pointing to a lower binding affinity of the non - phosphorylated rdx - gst to prg-2 ( see also figure s6p ) . we have therefore performed ips from stable prg-2-expressing hek293 cells transfected either with wt rdx - gfp ( rdx ) or non - phosphorylated rdx - gfp ( rdx ) using the described prg-2 antibody . in line with data from the peptide - microarray - based mapping , although binding of rdx was present at lower levels when compared to rdx , prg-2/rdx binding could be clearly detected ( figures 5p and 5q ) . this points to the fact that rdx phosphorylation , although not a prerequisite for rdx / prg-2 interaction , significantly increases rdx / prg-2 interaction . to answer the question of whether rdx / prg-2 association at the membrane is important for lpa - dependent rdx phosphorylation in neurons , we analyzed the effect of lpa stimulation on perm expression in wt and prg-2 neurons . here , lpa stimulation of wt neurons resulted in a significant increase in perm levels , while prg-2 neurons failed to show any significant change ( figures 5r and 5s ) . these results point to a yet unknown , but critical role of prg-2 in lpa - induced rdx activation . rdx is concentrated below the plasma membrane , a critical position linking transmembrane signaling to the actin cytoskeleton . since phosphorylation influences rdx activity in connecting the actin - cytoskeleton with the membrane ( fehon et al . , 2010 ) , transmembrane signaling events leading to rdx activation are optimally suited to act in extracellular cue mediated axon guidance . we therefore analyzed the effect of extracellular lpa in inducing perm changes at the gc membrane in wt and prg-2 neurons . in wt gcs , perm was concentrated in a compartment directly adjacent to the gc membrane ( figures 6a and 6b ) and strongly increased upon lpa stimulation ( figures 6e , 6f , and 6i ) . in contrast , no change of perm was observed in prg-2 gcs ( figures 6c , 6d , and 6g6i ) . moreover , direct comparison of perm levels in the center of the gc and in the periphery revealed a shift of the perm signal toward the membrane of wt gcs , but not of prg-2 gcs ( figure 6j ) . to prove whether rdx is dynamically recruited to the membrane via prg-2 binding induced by a local extracellular lpa source , we transfected wt or prg-2 neurons with gfp - tagged rdx and performed live imaging studies . while in wt neurons , local lpa stimulation rapidly induced a translocation of rdx - gfp to the membrane next to the lpa source ( figures 6k and 6l ) , prg-2 neurons did not show such a change ( figures 6 m and 6n ) . in sum , these data show that prg-2 is the mediator of lpa - induced localization of rdx to the gc membrane , where it is present in its phosphorylated form , in turn increasing binding to prg-2 . to prove that erm phosphorylation and activation play a role in lpa - induced gc turning , we analyzed the subcellular localization of perm in gcs of thalamic explants exposed to an lpa - rich environment ( 1 m lpa ) , as shown in figure 4i . detailed analysis of turning gcs , exemplarily encircled in figure 7a , revealed strong perm signal co - localized with f - actin at the tip of gcs turning away from the lpa - rich zone ( figures 7b7e ) . to confirm that prg-2 interacts with rdx at the gc , we performed a proximity ligation assay ( pla ) , a method that allows the study of in situ protein - protein interactions at resolutions < 40 nm ( rhett et al . , 2011 ) . here , we found a strong signal at the gc tip , confirming the prg-2/rdx interaction at the site of lpa action ( figures 7f and 7 g ) . taken together , these results confirm the critical role of prg-2/rdx interaction for the induction of rdx activation in gcs by extracellular lpa , which eventually leads to axon turning . to further substantiate the role of rdx in lpa - induced gc turning , we analyzed the thalamocortical projection in rdx mice . using biocytin tracing in living e17 thalamocortical slices , we observed misrouted thalamocortical axons in rdx slices that aberrantly invaded the cp ( figures 7i7k),while in slices from control littermates , the thalamocortical projection was confined to the iz ( figures 7h and 7k ) . in order to better understand the functional role of the prg-2/rdx interaction in thalamocortical axon guidance , we analyzed the effect of partial reduction of these proteins in transheterozygous mice ( prg-2/rdx ) in thalamocortical slices at e17 . here , the thalamocortical projection aberrantly invaded the cp , suggesting that it is indeed the interaction of prg-2 and rdx that is important for correct guidance of these fibers by an lpa gradient ( figures 7l7o ) . these data corroborate the idea that the lpa - prg-2-rdx / perm signal transduction axis is critical for correct guidance and restriction of thalamocortical axons to the iz . moreover , in line with decreased rdx expression after e16 ( figures s6 g and s6h ) , these data could explain how thalamocortical axons are finally able to enter into their target cortical region . to assess the functional outcome of altered thalamocortical projections in prg-2 mice , we first measured intrinsic optical responses in the corresponding barrel upon single - whisker back - and - forth deflection . this approach measures blood - flow changes in the representation of single whiskers in the barrel cortex upon activation . using this method , we found that the response area upon single - whisker stimulation , which corresponds to the stimulated barrel ( for morphological barrel assessment , see figure s7a ) , was larger in prg-2 mice . these data point to a more diffuse and broader cortical response extending beyond the border of the activated barrel ( figures 8a8c ) . moreover , it reflects our findings using retrograde tracing of thalamocortical fibers in prg-2 mice showing a clear overlap in the termination pattern of neurons from neighboring thalamic barreloids in the vpm ( figures 1r , 1 t , and 1u ) . this is in agreement with enlargement of functional barrel columns in mice due to impaired precision of the thalamocortical projection , as reported in other mouse models ( lokmane et al . , 2013 ) . to get a layer - specific resolution of neuronal activity , we performed in vivo electrophysiological recordings using multi - channel electrodes . we observed in prg-2 mice a slight decrease in multi - unit activity ( mua ) upon single - whisker deflection in layer iv and a significant decrease in layers ii / iii for prg-2 mice compared to wt controls ( figures 8d8f ) , which are the main target layers of the thalamocortical projection ( meyer et al . recent data suggest that layers ii / iii contain neurons specifically responsive for texture coarseness ( garion et al . , 2014 ) . we therefore assessed the ability of prg-2 mice to distinguish texture coarseness using a modified radial arm maze , where arms were covered with sandpaper of two slightly different grades ( 60 grade [ grain size , 265 m ] and 80 grade [ grain size , 190 m ] ; figure 8 g ) . only arms covered with the coarser ( 60 grade ) sandpaper were rewarded by accessible food pellets and counted as correct entrance . since experiments were performed in darkness and no other cues were presented , mice had to orient by sensing sandpaper roughness . while wt mice showed a significant improvement in their ability to choose the correct arm , prg-2 mice did not improve beyond chance levels even after 9 days of continuous training ( figure 8h ) . to exclude bias by an impaired learning ability , we tested prg-2 mice in the morris water maze ( mwm ) . escape latency for visible and hidden platforms was indistinguishable between prg-2 and wt littermates ( figures s7e was not impaired in prg-2 mice , we conclude that their performance at chance level in the texture discrimination task is a direct result of impaired thalamocortical axon projection precision . in line with unaltered learning ability , motor performance was also unaltered , as shown by analysis in the mwm or supporting limb responses ( figures s7h s7k ) . to exclude an effect of prg-2 deletion outside the thalamus , e.g. , in the cortex , which could affect cortical processing of somatosensory information , we also tested the above described thalamus - specific prg-2-deficient mouse line ( prg-2 ) . as shown in figures s8l s8o using retrograde tracing of thalamocortical fibers projecting to the cortical barrels , these animals display a similar phenotype as described for the constitutive prg-2 animals , where we could show that different barrels were aberrantly innervated by the same neuron in the thalamic vpm . furthermore , staining for prg-2 revealed that fibers originating from gbx-2-expressing thalamic neurons ( identified by the rfp reporter ) did not express prg-2 ( figures s7p s7s ) . behavioral analysis of the prg-2 mice in the sandpaper radial maze revealed that these mice displayed the same phenotype as the constitutive prg-2 mice and were not able to distinguish between the two sandpapers of different coarseness ( figure 8h ) . together with our in vivo imaging and electrophysiological data , these findings strongly indicate that misguided thalamocortical axons in prg-2 mice alter cortical information processing , resulting in a specific deficit in sensory discrimination at adult ages . an intriguing phenomenon in cortical development is the specific outgrowth of thalamocortical fibers that reach the appropriate cortical area before their target cells migrate to their final position in the cp ( kostovic and rakic , 1990 ) . as shown in other systems , thalamocortical axons accumulate in the iz below the cp ( ghosh et al . , , they interact with a transient neuronal population , the subplate neurons , which keep them in place and are thereby critically important for correct patterning of the thalamocortical connections . although the role of subplate neurons has long been recognized and gene expression patterns of subplate neurons have been described ( for review , see hoerder - suabedissen and molnr , 2015 ) , molecular mechanisms regulating the subplate neurons / thalamocortical fiber interaction are far from being understood . here , we analyzed the role of prg-2/lppr3 , a phospholipid - interacting molecule strongly expressed in thalamocortical axons during development ( figure 1a ) . using a constitutive prg-2 mouse line as well as a thalamus - specific prg-2 ko line ( prg-2/gbx2/r26 ) , where tamoxifen application at e12.5 leads to specific deletion of prg-2 in the thalamus ( prg-2 ; normand et al . , 2013 ) , we discovered that prg-2-deficient thalamocortical axons prematurely and aberrantly entered the cp . this altered the precise somatotopic termination of thalamocortical fibers in the somatosensory barrel field cortex and led to a deficit in sensory discrimination . molecular analysis revealed a signal transduction axis in the gc leading from the lpa - synthesizing molecule atx to lpa , prg-2 , and finally to phosphorylated rdx , a well - described linker between the gc membrane and the actin cytoskeleton . we demonstrate this atx / lpa / prg-2/prdx signaling axis on thalamocortical axon gc turning and suggest that this mechanism is responsible for keeping thalamocortical axons in the iz before entering the cp . the role of subplate cells was concluded from ablation experiments , where thalamocortical fibers , no longer able to contact subplate cells , escaped the subcortical waiting period and extended beyond their target cortical regions . this specific phenotype relies mechanistically on the lost sensitivity of thalamocortical axons toward extracellular lpa gradients synthesized by atx , a diffusible molecule expressed in subplate neurons ( hoerder - suabedissen et al . , 2013 ) . eventually , this leads to loss of proper axon guidance and to an invasion of inappropriate cortical compartments . since we also analyzed thalamus - specific prg-2 ko animals ( normand et al . , 2013 ) , we could rule out that this misrouting is due to defects , e.g. , derived from cortical prg-2 deficiency . interestingly , the same phenotype was detected when the catalytic function of atx was blocked , thereby impeding lpa production . our data suggest a thalamocortical guidance mechanism with a mutual dependence on lpa / prg-2 interaction . the critical importance of prg-2-mediated extracellular lpa effects on thalamic axon guidance was further clarified by single - axon analysis . while downstream mechanisms mediating lpa gc collapse have been described ( campbell and holt , 2003 ) , direct and specific lpa - interacting molecules involved in this mechanism remain unclear . in line with live imaging data , wt axons were repelled / collapsed when in contact with 10 m lpa , while lower lpa concentrations ( 1 m ) induced a turning behavior in gcs . however , prg-2 axons were not affected even by high lpa concentrations ( 10 m ) . searching for downstream interacting partners , we found rdx to directly interact and be co - expressed with prg-2 in thalamocortical axons . moreover , subcellular analyses revealed rdx to interact with prg-2 in gcs as shown by pla . importantly , ip experiments revealed that the prg-2/rdx binding was highly increased upon lpa stimulation . the role of this prg-2/rdx interaction was functionally confirmed , showing that prg-2 is critically important for rdx activation by extracellular lpa . while in wt neurons , concentrations as low as 100 nm lpa induced clear rdx phosphorylation , in prg-2 neurons , rdx phosphorylation did not increase , even upon 15 min of 1 m lpa stimulation . our data showing rdx phosphorylation upon lpa stimulation in full - length prg-2-expressing hek293 cells suggest that prg-2/rdx association increased rdx phosphorylation upon lpa stimulation . however , when analyzing binding to prg-2 to either wt rdx - gfp or its non - phosphorylated form ( rdx - gfp ) , we detected binding under both conditions , albeit with a significant reduction of binding to prg-2 of the non - phosphorylated form . these data show that rdx phosphorylation is not a prerequisite for prg-2/rdx binding ; however , it influences this binding . our finding that transheterozygous loss of prg-2 and rdx already results in altered thalamocortical fiber projection indicates an important role of the lpa / prg-2/rdx signal axis . since the active form of erms ( perm ) is critical for gc guidance ( mintz et al . , 2008 ) , we investigated the role of this signal axis at the gc , the critical location for axon guidance . here , lpa stimulation induced strong erm phosphorylation at the gc membrane of wt axons , while prg-2 gcs failed to show these changes . in line , rdx was rapidly translocated to the membrane directly next to a local lpa source , indicating an active role of lpa / prg-2/rdx signaling in gc guidance . together with the detection of perm adjacent to f - actin at the tip of turning gcs , this signaling axis appears to directly control lpa - induced axon turning . using in vivo imaging and high - density multi - electrode recordings in the barrel cortex of prg-2 mice and performing behavioral tests both in prg-2 mice and prg-2 animals , we elucidated the functional consequences of prg-2 deficiency and subsequent altered thalamocortical connectivity in cortical layers iv and ii / iii as well as in the characteristic discrimination abilities associated with these layers ( garion et al . , 2014 ) . it appears that rather specific deficits can be observed even upon drastic developmental defects resulting in grossly impaired cortical lamination and subsequent changes in the thalamocortical projection ( wagener et al . , 2016 ) , as shown by preserved functional sensory maps in the somatosensory barrel cortex observed in reelin - deficient mice ( guy et al . , 2015 ) . ( 2015 ) suggest microcircuits in layers ii / iii are important for specific stimulus orientation ( cossell et al . , 2015 ) , supporting the idea that layer ii / iii neurons mediate rather specific functions . this implies that functional changes in layers ii / iii seen in prg-2 mice can best explain the sensory discrimination deficits of the animal . taken together , we describe a novel molecular guidance mechanism involving phospholipid signaling influencing thalamocortial axon guidance at a critical time point , at which axons exhibit a waiting period at the cortical subplate . the alteration of the lpa / prg-2/prdx signal transduction pathway resulted in distinct electrophysiological and behavioral deficits in adult animals and highlighted the importance of this pathway for proper thalamocortical connectivity . all experiments were conducted in accordance with the national laws for the use of animals in research and with the european communities council directive 86/609/eec , and approved by the local ethical committee ( landesuntersuchungsamt rheinland - pfalz 23 . details of the generation and breeding of prg-2 , prg-2/gbx2/r26 , and rdx mice are provided in the supplemental experimental procedures . immunofluorescent ( if ) stainings , pla for detection of in situ protein interaction , and anterograde and retrograde tracing experiments were performed following standard protocols . organotypic slices were prepared as described in the supplemental experimental procedures . for biocytin tracing experiments , pressure injections were performed into e17 embryonic slices using glass capillaries attached to a toohey spritzer ( toohey company ) to induce biocytin alexa fluor 488 ( thermo fisher scientific ) . slices were then incubated for 6 hr and fixed with 4c pfa ( 4% ) . for electroporation experiments , corresponding plasmids were injected into organotypic slices from e15.5 brains that were electroporated by needle tungsten electrodes and an nepa21 super electroporator ( nepa gene ) . . , 2012 ) , cultivated for 2 days in vitro ( div ) and used for assessment of perm levels in the gc . if staining and perm assessment were performed as described in the supplemental experimental procedures . ip studies were performed on e17 brain material or on cell lysate from stably prg-2-expressing hek293 cells according to standard protocols . intrinsic signal optical imaging ( ioi ) and in vivo electrophysiological recordings in the barrel cortex of adult mice were performed according to standard protocols as described in the supplemental experimental procedures . sandpaper discrimination test and mwm were performed according to standard procedures . for animal experiments , mice from the same litter or of similar age following experiments , results were analyzed , animals were re - genotyped , and corresponding final statistical analyses were performed . briefly , after assessing for normal distribution ( using the kolmogorov - smirnov test ) , comparison between two groups , if not otherwise stated , was performed using a two - tailed unpaired t test for normal distributed data or a mann - whitney test for nonparametric data . when data were normalized to controls ( set as 100% ) , a one - sample t test was used . for quantitative assessment of aberrant fibers in traced or electroporated slices , a fisher s exact test was applied . comparison between more than two groups was performed using a one - way anova for parametric data or a kruskal - wallis test for nonparamatric data . variance was similar between groups and was estimated using either the f - test or the brown - forsythe test and bartlett s test . post hoc analysis for more than two groups for parametric data was performed using the bonferroni adjustment for multiple comparisons and dunn s test for nonparametric data . for behavioral analyses assessing genotype effects and time effects , as well as for comparisons of the multiple binding sites of prg-2 , h.j . , n.s . , h.m.m . , a.h .- s . , p .- h.p . , w.f . , and t.t . provided reagents ; writing original draft , j.v . ; review and editing , j.v . , r.n . , j.c .

epidemiological studies have suggested that interleukin-17 ( il-17 ) polymorphisms are associated with cancer risk . however , the results of these studies are inconsistent . therefore , we performed a meta - analysis to obtain a precise conclusion . odds ratios ( ors ) with 95% confidence intervals ( cis ) were used to assess the association of the il-17a rs2275913g > a and il-17f rs763780t > c polymorphisms with cancer risk . publication bias and sensitivity analyses were performed to ensure the statistical power . overall , 10 relevant case - control studies involving 4,516 cases and 5,645 controls were included . the pooled ors with 95% cis indicated that the il-17a rs2275913g > a polymorphism was significantly associated with increased cancer risk ( for a versus g : or = 1.28 , 95% ci : 1.161.41 , p < 0.001 , i2 = 61.1% ; for ga versus gg : or = 1.12 , 95% ci : 1.021.23 , p = 0.015 , i2 = 27.8% ; for aa versus gg : or = 1.71 , 95% ci : 1.382.41 , p < 0.001 , i2 = 69.6% ; for ga + aa versus gg : or = 1.23 , 95% ci : 1.131.34 , p < 0.001 , i2 = 6.4% ; for aa versus gg + ga : or = 1.62 , 95% ci : 1.272.07 , p < 0.001 , i2 = 81.4% ) . succeeding analysis of hwe and stratified analysis of gastric cancer and the asian ( and chinese ) population revealed similar results . the il-17f rs763780t > c polymorphism was also significantly associated with gastric cancer development . overall , the present meta - analysis suggests that il-17 polymorphisms increase the risk of developing cancer , particularly gastric cancer , in the asian ( and chinese ) population . summaryprecise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information . here , we show that prg-2 , a phospholipid - interacting molecule , is important for thalamocortical axon guidance . developing thalamocortical fibers both in prg-2 full knockout ( ko ) and in thalamus - specific ko mice prematurely entered the cortical plate , eventually innervating non - corresponding barrels . this misrouting relied on lost axonal sensitivity toward lysophosphatidic acid ( lpa ) , which failed to repel prg-2-deficient thalamocortical fibers . prg-2 electroporation in the prg-2/ thalamus restored the aberrant cortical innervation . we identified radixin as a prg-2 interaction partner and showed that radixin accumulation in growth cones and its lpa - dependent phosphorylation depend on its binding to specific regions within the c - terminal region of prg-2 . in vivo recordings and whisker - specific behavioral tests demonstrated sensory discrimination deficits in prg-2/ animals . our data show that bioactive phospholipids and prg-2 are critical for guiding thalamic axons to their proper cortical targets .

vitamin a deficiency is the leading cause of preventable childhood blindness in the developing world . although rare in the united states , vitamin a deficiency has been known to occur as a result of poor dietary intake , liver diseases , and gastrointestinal malabsorption [ 2 , 3 , 4 , 5 ] . vitamin a is a fat - soluble vitamin ingested in the diet in two forms : as retinol itself from animal sources , such as milk , meat , fish , liver , and eggs , or as the provitamin carotene from plant sources , such as green leafy vegetables , yellow fruits , and red palm oil . on the ocular surface , vitamin a deficiency has a wide range of ocular manifestations including conjunctival and corneal xerosis , keratomalacia , retinopathy , visual loss , and nyctalopia , also called night blindness , which is the earliest and most common symptom . we report a case of bilateral sequential corneal ulceration in a patient with severe vitamin a deficiency in the context of eosinophilic gastroenteropathy . a 29-year - old man was referred to our cornea unit with a left corneal ulcer of 6 weeks ' duration . he was previously diagnosed with eosinophilic gastroenteropathy ( biopsy proven ) , skin atopy and atopic keratoconjunctivitis . he had been treated with peroral steroids 15 mg / day and methotrexate 6 mg / week since the age of 11 years for his gastroenteropathy . he had surgery for steroid - induced cataracts with toric intraocular lens implantation at the age of 27 years . in addition , he had a number of food intolerances and subsisted mostly on a diet of potatoes . prior to his presentation , he had noted irritation , itchiness , and a decrease in vision in his left eye for 6 weeks and was treated with topical anti - allergic and topical steroid drops ; however , no improvement could be observed . polymerase chain reaction testing for herpes simplex virus was negative , and the cultures did not show any growth . acuity without correction was 20/80 od , improved by pinhole to 20/50 , and 20/150 os with a manifest refraction of 0.25/-2.5/5 , not improved by pinhole . the intraocular pressure was 19 mm hg od and 21 mm hg os by tonopen . slit - lamp examination showed temporal wrinkling of the conjunctiva in both eyes , with a dry granular pattern and loss of transparency ( fig . the left temporal cornea showed an epithelial defect overlying a partial - thickness ulcer with approximately 50% thinning and adjacent limbitis . in both eyes , the anterior chambers were quiet and there were well - centered posterior chamber iols . our patient was initially diagnosed with peripheral ulcerative keratitis in the left eye in the presence of atopic keratoconjunctivitis . his steroids were increased to 50 mg / day and tapered every 3 days by 10 mg / day . he was put on preservative - free artificial tears . a vasculitic workup ( c - reactive protein , erythrocyte sedimentation rate , antinuclear antibody , antineutrophil cytoplasmic antibody , rheumatoid factor , syphilis serology ) was unremarkable . serum was sent for analysis of vitamin a and retinol - binding protein ( rbp ) levels , with a processing time of several weeks at our location . the ulcer in the left eye epithelialized over the following 2 weeks and subsequently vascularized from the temporal conjunctiva . two weeks later , the patient presented to our clinic again with significant right eye pain . he had a corneal ulcer in his right eye , complicated by a small corneal perforation , with the iris plugging the wound ( fig . serendipitously , at that time , the results of his serum vitamin a levels were available , revealing that the levels were significantly decreased to 0.2 mol / l ( normal range 0.9 - 2.5 ) . the rbp level was also reduced to 12 mg / l ( normal 30 - 92 ) . the patient was treated with iv multivitamin ( containing 10,000 iu vitamin a , c , d , e and b complex ) for 5 consecutive days while in hospital , and then with oral vitamin a 200,000 iu once a week for 2 weeks . 2b ) and the corneal keratinization in both eyes resolved over the following 6 weeks ( fig . we present a rare case of vitamin a deficiency secondary to a malnutrition syndrome leading to bilateral xerosis and sequential corneal ulceration . similar reports from developed populations are rare , but have been described in association with disorders such as chronic alcoholism , cystic fibrosis , and anorexia nervosa . the initial treatment with an increase of systemic steroids improved the ulceration and seemed to suggest an immunogenic process . however , the involvement of the second eye while on steroid treatment forced consideration of other causes . clinical suspicion of xerophthalmia at the time of presentation prompted testing for vitamin a and serum rbp . it was unfortunate in our case that the progression of the disease occurred in the time taken for results to be reported . the earliest ocular symptom of vitamin a deficiency is nyctalopia , secondary to depletion of photopigments . unfortunately , we were not able to obtain an electroretinogram ( erg ) before the treatment . an abnormal erg at presentation in association with the anterior segment findings would have helped to establish a diagnosis of vitamin a deficiency before serum levels were available . it has been shown that eosinophilic gastroenteropathy can cause malabsorption or protein - losing enteropathy [ 11 , 12 ] , but in our case , a gastroenterology consult confirmed inactive disease and showed no other signs of malabsorption ( normal albumin levels and international normalized ratio ) . the most likely reason was felt to be malnutrition due to lack of vitamin a uptake secondary to the patient 's multiple food intolerances that accompany eosinophilic gastroenteropathy . the long - lasting success of oral vitamin a supplementation further favors previous malnutrition over malabsorption . the oral dosage regimen is 200,000 iu vitamin a in oil , followed the next day with an additional dose of 200,000 iu . if patients have severe corneal disease or malabsorption , the preferred dose is 100,000 iu water - miscible vitamin a administered intramuscularly . it is important to note that if vitamin a deficiency is associated with a protein - energy malnutrition syndrome , protein levels must be corrected at the same time as vitamin supplementation . our case serves as a reminder to corneal specialists in developed populations of the potentially devastating effects of vitamin a deficiency on the eye . vitamin a deficiency should be suspected in any practice in the presence of corneal ulceration in combination with malabsorption and malnutrition syndromes . grounding or earthing refers to direct skin contact with the surface of the earth , such as with bare feet or hands , or with various grounding systems . subjective reports that walking barefoot on the earth enhances health and provides feelings of well - being can be found in the literature and practices of diverse cultures from around the world.1 for a variety of reasons , many individuals are reluctant to walk outside barefoot , unless they are on holiday at the beach . various grounding systems are available that enable frequent contact with the earth , such as while sleeping , sitting at a computer , or walking outdoors . these are simple conductive systems in the form of sheets , mats , wrist or ankle bands , adhesive patches that can be used inside the home or office , and footwear . these applications are connected to the earth via a cord inserted into a grounded wall outlet or attached to a ground rod placed in the soil outside below a window . for the footwear applications , a conductive plug is positioned in the shoe sole at the ball of the foot , under the metatarsals , at the acupuncture point known as kidney 1 . from a practical standpoint , these methods offer a convenient and routine , user - friendly approach to grounding or earthing . they can also be used in clinical situations , as will be described in the section entitled summary of findings to date.1 recently , a group of about a dozen researchers ( including the authors of this paper ) has been studying the physiological effects of grounding from a variety of perspectives . this research has led to more than a dozen studies published in peer - reviewed journals . while most of these pilot studies involved relatively few subjects , taken together , the research has opened a new and promising frontier in inflammation research , with broad implications for prevention and public health . the findings merit consideration by the inflammation research community , which has the means to verify , refute , or clarify the interpretations we have made thus far . grounding reduces or even prevents the cardinal signs of inflammation following injury : redness , heat , swelling , pain , and loss of function ( figures 1 and 2 ) . rapid resolution of painful chronic inflammation was confirmed in 20 case studies using medical infrared imaging ( figure 3).2,3 our main hypothesis is that connecting the body to the earth enables free electrons from the earth s surface to spread over and into the body , where they can have antioxidant effects . specifically , we suggest that mobile electrons create an antioxidant microenvironment around the injury repair field , slowing or preventing reactive oxygen species ( ros ) delivered by the oxidative burst from causing collateral damage to healthy tissue , and preventing or reducing the formation of the so - called inflammatory barricade . we also hypothesize that electrons from the earth can prevent or resolve so - called silent or smoldering inflammation . if verified , these concepts may help us better understand and research the inflammatory response and wound healing , and develop new information on how the immune system functions in health and disease . grounding appears to improve sleep , normalize the day night cortisol rhythm , reduce pain , reduce stress , shift the autonomic nervous system from sympathetic toward parasympathetic activation , increase heart rate variability , speed wound healing , and reduce blood viscosity . a summary has been published in the journal of environmental and public health.4 one of the first published grounding studies examined the effects of grounding on sleep and circadian cortisol profiles.5 the study involved 12 subjects who were in pain and had problems sleeping . they slept grounded for 8 weeks using the system shown in figure 4 . during this period , their diurnal cortisol profiles normalized , and most of the subjects reported that their sleep improved and their pain and stress levels declined . the results of the experiment led to these conclusions : 1 ) grounding the body during sleep yields quantifiable changes in diurnal or circadian cortisol secretion levels that , in turn , 2 ) produce changes in sleep , pain , and stress ( anxiety , depression , and irritability ) , as measured by subjective reporting . the cortisol effects described by ghaly and teplitz5 are particularly significant in the light of recent research showing that prolonged chronic stress results in glucocorticoid receptor resistance.6 such resistance results in failure to downregulate inflammatory responses , which can thereby increase risks of a variety of chronic diseases . this effect complements the findings described in the effects on pain and the immune response section . a pilot study on the effects of grounding on pain and the immune response to injury employed delayed - onset muscle soreness ( doms).7 doms is the muscular pain and stiffness that takes place hours to days after strenuous and unfamiliar exercise . the phase of contraction that occurs when a muscle shortens , as in lifting a dumbbell , is referred to as concentric , whereas the phase of contraction as a muscle lengthens , as in lowering a dumbbell , is referred to as eccentric . eight healthy subjects performed an unfamiliar , eccentric exercise that led to pain in their gastrocnemius muscles . this was done by having them perform two sets of 20 toe raises with a barbell on their shoulders and the balls of their feet on a 2-inch 4-inch wooden board.7 all subjects ate standardized meals at the same time of day , and adhered to the same sleep cycle for 3 days . at 5.40 pm on each day , four of the subjects had conductive grounding patches adhered to their gastrocnemius muscles and the bottoms of their feet . they remained on the grounded sheets except for visits to the bathroom and meals . as controls , four subjects followed the same protocol except that their patches and sheets were not grounded . the following measurements were taken before the exercise and 1 , 2 , and 3 days thereafter : pain levels , magnetic resonance imaging , spectroscopy , cortisol in serum and saliva , blood and enzyme chemistry , and blood cell counts.7 pain was monitored with two techniques . the subjective method involved morning and afternoon use of a visual analog scale . in the afternoon , a blood pressure cuff was positioned on the right gastrocnemius and inflated to the point of acute discomfort . the grounded subjects experienced less pain , as revealed with both the analog soreness scale ( figure 5 ) and by their ability to tolerate a higher pressure from the blood pressure cuff ( figure 6).7 the doms grounding study report7 contains a summary of the literature on the changes in blood chemistry and content of formed elements ( erythrocytes , leukocytes , and platelets ) expected after an injury . the immune system detects pathogens and tissue damage and responds by initiating the inflammation cascade , sending neutrophils and lymphocytes into the region.812 as expected , the white cell counts increased in the ungrounded or control subjects . white cell counts in the grounded subjects steadily decreased following the injury ( figure 7).7 previous research has shown increases in neutrophils following injury.1316 this happened in both grounded and ungrounded subjects ( figure 8) , although neutrophil counts were always lower in the grounded subjects.7 as the number of neutrophils increases , lymphocytes are expected to decrease.1719 in the doms study , the lymphocyte count in the grounded subjects was always below the ungrounded subjects ( figure 9).7 normally , neutrophils rapidly invade an injured region8,2022 in order to break down damaged cells and send signals through the cytokine network to regulate the repair process . oxidative burst.21 while ros clear pathogens and cellular debris so that the tissue can regenerate , ros can also damage healthy cells adjacent to the repair field , causing so - called collateral damage . the fact that the grounded subjects had fewer circulating neutrophils and lymphocytes could indicate that the original damage resolved more quickly , collateral damage reduced , and the recovery process accelerated . this would explain the reduction in the cardinal signs of inflammation ( redness , heat , swelling , pain , and loss of function ) following acute injury , as documented , for example , in figures 1 and 2 , and the rapid reduction of chronic inflammation documented in figure 3 . our working hypothesis features this scenario : mobile electrons from the earth enter the body and act as natural antioxidants;3 they are semi - conducted through the connective tissue matrix , including through the inflammatory barricade if one is present;23 they neutralize ros and other oxidants in the repair field ; and they protect healthy tissue from damage . the fact that there are fewer circulating neutrophils and lymphocytes in the grounded subjects may be advantageous because of the harmful role these cells are thought to play in prolonging inflammation.24 we also raise the possibility that the inflammatory barricade is actually formed in ungrounded subjects by collateral damage to healthy tissue , as was suggested by selye in the first and subsequent editions of his book the stress of life ( figure 10).25 while there may be other explanations , we suggest that rapid resolution of inflammation takes place because the earth s surface is an abundant source of excited and mobile electrons , as described in our other work.1 we further propose that skin contact with the surface of the earth allows earth s electrons to spread over the skin surface and into the body . the meridians are known to be low resistance pathways for the flow of electrical currents.2628 another pathway is via mucous membranes of the respiratory and digestive tracts , which are continuous with the skin surface . sokal and sokal29 found that the electrical potential on the body , on the mucosal membrane of the tongue , and in the venous blood rapidly drop to approximately 200 mv . these effects reveal changes in the internal electrical environment within the body.29 selye30 studied the histology of the wall of the inflammatory pouch or barricade ( figure 10 ) . our hypothesis is that electrons can be semi - conducted across the barrier , and can then neutralize reactive oxygen species ( free radicals).30 a semiconducting collagen pathway or corridor may explain how electrons from the earth quickly attenuate chronic inflammation not resolved by dietary antioxidants or by standard medical care , including physical therapy ( figure 3 ) . taken together , these observations indicate that grounding or earthing the human body significantly alters the inflammatory response to an injury . one of the first published grounding studies examined the effects of grounding on sleep and circadian cortisol profiles.5 the study involved 12 subjects who were in pain and had problems sleeping . they slept grounded for 8 weeks using the system shown in figure 4 . during this period , their diurnal cortisol profiles normalized , and most of the subjects reported that their sleep improved and their pain and stress levels declined . the results of the experiment led to these conclusions : 1 ) grounding the body during sleep yields quantifiable changes in diurnal or circadian cortisol secretion levels that , in turn , 2 ) produce changes in sleep , pain , and stress ( anxiety , depression , and irritability ) , as measured by subjective reporting . the cortisol effects described by ghaly and teplitz5 are particularly significant in the light of recent research showing that prolonged chronic stress results in glucocorticoid receptor resistance.6 such resistance results in failure to downregulate inflammatory responses , which can thereby increase risks of a variety of chronic diseases . this effect complements the findings described in the effects on pain and the immune response section . a pilot study on the effects of grounding on pain and the immune response to injury employed delayed - onset muscle soreness ( doms).7 doms is the muscular pain and stiffness that takes place hours to days after strenuous and unfamiliar exercise . the phase of contraction that occurs when a muscle shortens , as in lifting a dumbbell , is referred to as concentric , whereas the phase of contraction as a muscle lengthens , as in lowering a dumbbell , is referred to as eccentric . eight healthy subjects performed an unfamiliar , eccentric exercise that led to pain in their gastrocnemius muscles . this was done by having them perform two sets of 20 toe raises with a barbell on their shoulders and the balls of their feet on a 2-inch 4-inch wooden board.7 all subjects ate standardized meals at the same time of day , and adhered to the same sleep cycle for 3 days . at 5.40 pm on each day , four of the subjects had conductive grounding patches adhered to their gastrocnemius muscles and the bottoms of their feet . as controls , four subjects followed the same protocol except that their patches and sheets were not grounded . the following measurements were taken before the exercise and 1 , 2 , and 3 days thereafter : pain levels , magnetic resonance imaging , spectroscopy , cortisol in serum and saliva , blood and enzyme chemistry , and blood cell counts.7 pain was monitored with two techniques . the subjective method involved morning and afternoon use of a visual analog scale . in the afternoon , a blood pressure cuff was positioned on the right gastrocnemius and inflated to the point of acute discomfort . the grounded subjects experienced less pain , as revealed with both the analog soreness scale ( figure 5 ) and by their ability to tolerate a higher pressure from the blood pressure cuff ( figure 6).7 the doms grounding study report7 contains a summary of the literature on the changes in blood chemistry and content of formed elements ( erythrocytes , leukocytes , and platelets ) expected after an injury . the immune system detects pathogens and tissue damage and responds by initiating the inflammation cascade , sending neutrophils and lymphocytes into the region.812 as expected , the white cell counts increased in the ungrounded or control subjects . white cell counts in the grounded subjects steadily decreased following the injury ( figure 7).7 previous research has shown increases in neutrophils following injury.1316 this happened in both grounded and ungrounded subjects ( figure 8) , although neutrophil counts were always lower in the grounded subjects.7 as the number of neutrophils increases , lymphocytes are expected to decrease.1719 in the doms study , the lymphocyte count in the grounded subjects was always below the ungrounded subjects ( figure 9).7 normally , neutrophils rapidly invade an injured region8,2022 in order to break down damaged cells and send signals through the cytokine network to regulate the repair process . oxidative burst.21 while ros clear pathogens and cellular debris so that the tissue can regenerate , ros can also damage healthy cells adjacent to the repair field , causing so - called collateral damage . the fact that the grounded subjects had fewer circulating neutrophils and lymphocytes could indicate that the original damage resolved more quickly , collateral damage reduced , and the recovery process accelerated . this would explain the reduction in the cardinal signs of inflammation ( redness , heat , swelling , pain , and loss of function ) following acute injury , as documented , for example , in figures 1 and 2 , and the rapid reduction of chronic inflammation documented in figure 3 . our working hypothesis features this scenario : mobile electrons from the earth enter the body and act as natural antioxidants;3 they are semi - conducted through the connective tissue matrix , including through the inflammatory barricade if one is present;23 they neutralize ros and other oxidants in the repair field ; and they protect healthy tissue from damage . the fact that there are fewer circulating neutrophils and lymphocytes in the grounded subjects may be advantageous because of the harmful role these cells are thought to play in prolonging inflammation.24 we also raise the possibility that the inflammatory barricade is actually formed in ungrounded subjects by collateral damage to healthy tissue , as was suggested by selye in the first and subsequent editions of his book the stress of life ( figure 10).25 while there may be other explanations , we suggest that rapid resolution of inflammation takes place because the earth s surface is an abundant source of excited and mobile electrons , as described in our other work.1 we further propose that skin contact with the surface of the earth allows earth s electrons to spread over the skin surface and into the body . the meridians are known to be low resistance pathways for the flow of electrical currents.2628 another pathway is via mucous membranes of the respiratory and digestive tracts , which are continuous with the skin surface . sokal and sokal29 found that the electrical potential on the body , on the mucosal membrane of the tongue , and in the venous blood rapidly drop to approximately 200 mv . these effects reveal changes in the internal electrical environment within the body.29 selye30 studied the histology of the wall of the inflammatory pouch or barricade ( figure 10 ) . our hypothesis is that electrons can be semi - conducted across the barrier , and can then neutralize reactive oxygen species ( free radicals).30 a semiconducting collagen pathway or corridor may explain how electrons from the earth quickly attenuate chronic inflammation not resolved by dietary antioxidants or by standard medical care , including physical therapy ( figure 3 ) . taken together , these observations indicate that grounding or earthing the human body significantly alters the inflammatory response to an injury . the concept that the inflammatory barricade forms from collateral damage to healthy tissue surrounding an injury site is supported by selye s classic studies published along with his description of the granuloma or selye pouch ( figure 10).25,30 moreover , research in cell biology and biophysics reveals the human body is equipped with a system - wide collagenous , liquid crystalline semiconductor network known as the living matrix,31 or in other terms , a ground regulation system32,33 or tissue tensegrity matrix system ( figure 11).34 this body - wide network can deliver mobile electrons to any part of the body and thereby routinely protect all cells , tissues , and organs from oxidative stress or in the event of injury.23,31 the living matrix includes the extracellular and connective tissue matrices as well as the cytoskeletons of all cells.31 integrins at cell surfaces are thought to allow for semi - conduction of electrons to the cell interior , and links across the nuclear envelope enable the nuclear matrix and genetic material to be part of the circuitry.23 our hypothesis is that this body - wide electronic circuit represents a primary antioxidant defense system . the extracellular part of the matrix system is composed mainly of collagen and ground substances ( figures 11 and 12 ) . this concept was introduced by albert szent - gyrgyi in the kornyi memorial lecture in budapest , hungary in 1941 . his talk was published in both science ( towards a new biochemistry?)35 and nature ( the study of energy levels in biochemistry).36 the idea that proteins might be semiconductors was immediately and firmly rejected by biochemists . many modern scientists continue to reject semi - conduction in proteins , because living systems only have trace amounts of silicone , germanium , and compounds of gallium that are the most widely used materials in electronic semiconductor devices . however , there are many ways of making organic semiconductors without using metals . one of the sources of confusion was the widely held belief that water was a mere filler material . we now know that water plays crucial roles in enzymatic activities and semi - conduction . hydrated proteins actually are semiconductors , and have become important components in the global microelectronics industry . organic microcircuits are preferred for some applications , because they can be made very small , self - assemble , are robust , and have low energy consumption.37,38 one of the leaders in the field of molecular electronics , ns hush , has recognized albert szent - gyrgyi and robert s mulliken for providing two concepts fundamental to the industrial applications : theories of biological semi - conduction , and molecular orbital theory , respectively.39 in recent studies , given awards by the materials research society in both europe and the usa , scientists from israel made flexible biodegradable semiconductor systems using proteins from human blood , milk , and mucus.40 silicon , the most widely used semiconducting material , is expensive in the pure form needed for semiconductors , and is inflexible and environmentally problematic . organic semiconductors are predicted to lead to a new range of flexible and biodegradable computer screens , cell phones , tablets , biosensors , and microprocessor chips . we have come a long way since the early days when semi - conduction in proteins was so thoroughly rejected.41,42,43 ground substance polyelectrolyte molecules associated with the collagenous connective tissue matrix are charge reservoirs ( figure 12 ) . the glycosaminoglycans have a high density of negative charges due to the sulfate and carboxylate groups on the uronic acid residues . the matrix is therefore a body - wide system capable of absorbing and donating electrons wherever they are needed to support immune functioning.44 the interiors of cells including the nuclear matrix and dna are all parts of this biophysical electrical storage and delivery system . the time - course of the effects of grounding on injury repair can be estimated in various ways . first , we know from medical infrared imaging that inflammation begins to subside within 30 minutes of connecting with the earth via a conductive patch placed on the skin.2,3 secondly , metabolic activity increases during this same period . specifically , there is an increase in oxygen consumption , pulse rate , and respiratory rate and a decrease in blood oxygenation during 40 minutes of grounding.45 we suspect that the filling of the charge reservoirs is a gradual process , possibly because of the enormous number of charged residues on the polyelectrolytes , and because they are located throughout the body . when charge reservoirs are saturated , the body is in a state we refer to as inflammatory preparedness this means that the ground substance , which pervades every part of the body , is ready to quickly deliver antioxidant electrons to any site of injury via the semiconducting collagenous matrix ( see figure 16b ) . these considerations also imply anti - aging effects of earthing or grounding , since the dominant theory of aging emphasizes cumulative damage caused by ros produced during normal metabolism or produced in response to pollutants , poisons , or injury.46 we hypothesize an anti - aging effect of grounding that is based on a living matrix reaching every part of the body and that is capable of delivering antioxidant electrons to sites where tissue integrity might be compromised by reactive oxidants from any source.47,48 molecules generated during the immune response were also followed in the doms study.7 parameters that differed consistently by 10% or more between grounded and ungrounded subjects , normalized to baseline , included creatine kinase , phosphocreatine / inorganic phosphate ratios , bilirubin , phosphorylcholine , and glycerolphosphorylcholine . bilirubin is a natural antioxidant that helps control ros.4953 while bilirubin levels decreased in both grounded and ungrounded groups , the margin between the subjects was large ( figure 13).7 the inflammatory markers changed at the same time that the pain indicators were changing . this was revealed by both the visual analog pain scale and by the pressure measurements on the right gastrocnemius ( figures 5 and 6 ) . the authors of the doms study suggested that bilirubin may have been used as a source of electrons in the ungrounded subjects.7 it is possible that the lower decline in circulating bilirubin in the grounded subjects was due to the availability in the repair field of free electrons from the earth . other markers encourage the hypothesis that the grounded subjects more efficiently resolved tissue damage : the pain measures , inorganic phosphate - phosphocreatine ratios ( pi / pcr ) , and creatine kinase ( ck ) . muscle damage has been widely correlated with ck.5456 as figure 14 shows , ck values in the ungrounded subjects were consistently above those in the grounded subjects.7 differences between pi / pcr of the two groups were monitored by magnetic resonance spectroscopy . these ratios are indicative of metabolic rate and cellular damage.5760 inorganic phosphate levels are indicative of hydrolysis of pcr and adenosine triphosphate . the ungrounded subjects had higher levels of pi , while the grounded subjects showed higher levels of pcr . these findings indicate that mitochondria in the grounded subjects are not producing as much metabolic energy , probably because there is less demand due to more rapid achievement of homeostasis . the pilot study7 on the effects of earthing in speeding recovery from the pain of doms provides a good basis for a larger study . the concepts presented here are summarized in figure 16 as a comparison between mr shoes ( an ungrounded individual ) and mr barefoot ( a grounded individual ) . a search for inflammation in the national library of medicine database ( pubmed ) reveals over 400,000 studies , with more than 34,000 published in 2013 alone . the most common cause of death and disability in the united states is chronic disease . seventy - five percent of the nation s health care spending , which surpassed us$2.3 trillion in 2008 , is for treating chronic disease . heart disease , cancer , stroke , chronic obstructive pulmonary disease , osteoporosis , and diabetes are the most common and costly chronic diseases.61 others include asthma , alzheimer s disease , bowel disorders , cirrhosis of the liver , cystic fibrosis , multiple sclerosis , arthritis , lupus , meningitis , and psoriasis . osteoporosis affects about 28 million aging americans.61,62 however , there are few theories on the mechanisms connecting chronic inflammation with chronic disease . the research on grounding or earthing summarized here provides a logical and testable theory based on a variety of evidence . the textbook description of the immune response describes how large or small injuries cause neutrophils and other white blood cells to deliver highly ros and rns to break down pathogens and damaged cells and tissues . classical textbook descriptions also refer to an inflammatory barricade that isolates injured tissues to hinder the movement of pathogens and debris from the damaged region into adjacent , healthy tissues . selye described how the debris coagulates to form the inflammatory barricade ( figure 10 ) . this barrier also hinders the movements of antioxidants and regenerative cells into the blocked - off area . repair can be incomplete , and this incomplete repair can set up a vicious inflammatory cycle that can persist for a long period of time , leading to so - called silent or smoldering inflammation that in turn , over time , can promote the development of chronic disease . remarkable as it may seem , our findings suggest that this classical picture of the inflammatory barricade may be a consequence of lack of grounding , and of a resultant electron deficiency . wounds heal very differently when the body is grounded ( figures 1 and 2 ) . healing is much faster , and the cardinal signs of inflammation are reduced or eliminated . those who research inflammation and wound healing need to be aware of the ways grounding can alter the time - course of inflammatory responses . they also need to be aware that the experimental animals they use for their studies may have very different immune systems and responses , depending on whether or not they were reared in grounded or ungrounded cages . it is standard research practice for investigators to carefully describe their methods and the strain of the animals they use so that others can repeat the studies if they wish . an assumption is that all wistar rats , for example , will be genetically and physiologically similar . however , a comparison of neoplasms in sprague dawley rats ( originally outbred from the wistar rat ) from different sources revealed highly significant differences in the incidences of endocrine and mammary tumors . the frequency of adrenal medulla tumors also varied in rats from the same suppliers raised in different laboratories . the authors stressed the need for extreme caution in evaluation of carcinogenicity studies conducted at different laboratories and/or on rats from different sources.63 from our perspective , these variations are not at all surprising . animals will differ widely in the degree to which their charge reservoirs are saturated with electrons . are their cages made of metal , and if they are , is that metal grounded ? how close are their cages to wires or conduits carrying 60/50 hz electricity ? from our research , those factors will have measurable impacts on immune responses . hidden variable that could have affected the outcomes of countless studies , and also could affect the ability of other investigators to reproduce a particular study . dominant lifestyle factors such as insulating footwear , high - rise buildings , and elevated beds separate most humans from direct skin connection with the earth s surface . an earth connection was an everyday reality in past cultures that used animal skins for footwear and to sleep on . we suggest that the process of killing pathogens and clearing debris from injury sites with ros and rns evolved to take advantage of the body s constant access to the virtually limitless source of mobile electrons the earth provides when we are in contact with it . antioxidants are electron donors , and the best electron donor , we strongly believe , is right under our feet : the surface of the earth , with its virtually unlimited storehouse of accessible electrons . electrons from the earth may in fact be the best antioxidants , with zero negative secondary effects , because our body evolved to use them over eons of physical contact with the ground . our immune systems work beautifully as long as electrons are available to balance the ros and reactive nitrogen species ( rns ) used when dealing with infection and tissue injury . our modern lifestyle has taken the body and the immune system by surprise by suddenly depriving it of its primordial electron source . this planetary separation began accelerating in the early 1950s with the advent of shoes made with insulating soles instead of the traditional leather . the disconnection from the earth may be an important , insidious , and overlooked contribution to physiological dysfunction and to the alarming global rise in non - communicable , inflammatory - related chronic diseases . a lack of electrons can also de - saturate the electron transport chains in mitochondria , leading to chronic fatigue and slowing the cellular migrations and other essential activities of the cells of the immune system.64 at this point , even a minor injury can lead to a long - term health issue . when mobile electrons are not available , the inflammatory process takes an abnormal course . cells of the immune system may fail to distinguish between the body s diverse chemical structures ( called self ) and the molecules of parasites , bacteria , fungi , and cancer cells ( called non - self ) . this loss of immunologic memory can lead to attacks by some immune cells on the body s own tissues and organs . an example is the destruction of insulin - producing beta cells of the islets of langerhans in the diabetic patient . lupus erythematosus is an extreme example of an auto - immune condition caused by the body s immune system attacking host tissues and organs . lupus , for example , can affect many different body systems , including skin , kidneys , blood cells , joints , heart , and lungs . with time , the immune system becomes weaker and the individual more vulnerable to inflammation or infections that may not heal , as often seen with the wounds of diabetic patients . specifically , which part or parts of the body the weakened immune system will attack first depends on many factors such as genetics , habits ( sleep , food , drinks , exercise , etc ) , and toxins in the body and in the environment.65,66 a repeated observation is that grounding , or earthing , reduces the pain in patients with lupus and other autoimmune disorders.1 accumulating experiences and research on earthing , or grounding , point to the emergence of a simple , natural , and accessible health strategy against chronic inflammation , warranting the serious attention of clinicians and researchers . the living matrix ( or ground regulation or tissue tensegrity - matrix system ) , the very fabric of the body , appears to serve as one of our primary antioxidant defense systems . as this report explains , it is a system requiring occasional recharging by conductive contact with the earth s surface

purpose : vitamin a deficiency is a very rare condition in the developed world and can lead to a variety of ocular changes from xerosis and xerophthalmia to corneal ulcer and perforation . the treatment of this devastating disease is simple and inexpensive . it is therefore important to recognize and treat accordingly , especially in the event of ulcers unresponsive to treatment or in the presence of severe malnutrition / malabsorption syndromes . the purpose of this case report is to remind physicians of the potentially devastating effects of vitamin a deficiency on the eyes and to demonstrate outcomes after vitamin a treatment . methods : single observational case report . results : a 29-year - old male with known eosinophilic gastroenteropathy was treated with oral steroids for peripheral ulcerative keratitis . two weeks after resolution , the patient suffered from peripheral ulcerative keratitis in his other eye , with a self - sealing perforation . vitamin a deficiency was confirmed and successfully treated , leading to subsequent resolution of signs and symptoms . conclusions : vitamin a deficiency can be present in patients with malabsorption and malnutrition syndromes and should be considered as cause of corneal ulceration . multi - disciplinary research has revealed that electrically conductive contact of the human body with the surface of the earth ( grounding or earthing ) produces intriguing effects on physiology and health . such effects relate to inflammation , immune responses , wound healing , and prevention and treatment of chronic inflammatory and autoimmune diseases . the purpose of this report is two - fold : to 1 ) inform researchers about what appears to be a new perspective to the study of inflammation , and 2 ) alert researchers that the length of time and degree ( resistance to ground ) of grounding of experimental animals is an important but usually overlooked factor that can influence outcomes of studies of inflammation , wound healing , and tumorigenesis . specifically , grounding an organism produces measurable differences in the concentrations of white blood cells , cytokines , and other molecules involved in the inflammatory response . we present several hypotheses to explain observed effects , based on current research results and our understanding of the electronic aspects of cell and tissue physiology , cell biology , biophysics , and biochemistry . an experimental injury to muscles , known as delayed onset muscle soreness , has been used to monitor the immune response under grounded versus ungrounded conditions . grounding reduces pain and alters the numbers of circulating neutrophils and lymphocytes , and also affects various circulating chemical factors related to inflammation .

food chain information ( fci ) is one of the innovations introduced by the hygiene package to enhance the concept of food security and ensure welfare and animal health . food chain information can be used by the operator of the slaughterhouse within the haccp plan in order to organise the slaughtering and by the official veterinarian to take decisions on the carcass . information should cover what follows ( european commission , 2004 ) : i ) status of provenance or the regional animal health status ; veterinary medicinal products or other treatments administered within a relevant period and with a withdrawal period > 0 , together with their dates of administration and withdrawal periods ; ii ) occurrence of diseases that may affect the safety of the meat ; iii ) the results , if they are relevant to the public health , of any analysis carried out on samples taken from the animals or other samples taken to diagnose diseases that may affect the safety of the meat , including samples taken in a framework of the monitoring and control of zoonoses and residues ; iv ) relevant reports about previous ante- and post - mortem inspections of animals from the same holding of provenience , including reports from the official veterinarian production data when they may indicate the presence of diseases ; v ) name and address of the private veterinarian normally attending the holding of provenience . fci must be provided to the slaughterhouse operators at least 24 h before the arrival of the animals . it is also possible to send fci simultaneously in the following cases : for pigs , poultry or farmed game that have passed the antemortem inspection at the farm and are accompanied by a veterinary certificate declaring the animals as healthy ; for slaughtering of domestic solipeds or other animals subject to emergency slaughter if accompanied by a veterinary certificate attesting the successful antemortem inspection ; for animals that have not been sent directly from the holding of provenance to the slaughterhouse . within the council regulation ( ec ) n. 2076/2005 ( european commission , 2005 ) there is another exception to regulation ( ec ) n. 853/2004 ( european commission , 2004 ) , which provides for the simultaneous transmission of fci and animals regardless of species , subject to agreements between food business operator ( fbo ) and the official veterinary . if the veterinary decides for the slaughtering , even in the absence of fci , the document must be provided within 24 h , otherwise the entire carcass will be destroyed . several eu member states drafted guidelines for the correct application of the document . unfortunately in italy only few regions did it . the aim of the present work is to compare the different european guidelines and to analyse the situation in piedmont after few years of the entry into force of this provision in order to assess the presence of potential problems and propose solutions . we analysed the guidelines of : france , united kingdom , belgium , the netherlands , spain and germany ( affsa , 2008 ; spanish government , 2009 ; afsca , 2009 ; pve , 2014 ) . for each country we evaluated the guidelines and compared information contained within the fci for the different animal species . in order to analyse the situation in the piedmont year we collected for one year and compared the fci in 11 slaughterhouses differing among them for capacity and slaughtered species ( cattle , pigs , small ruminants , poultry , rabbits and horses ) . the fci models were classified as : fci negative ( compiled form with one or more items not completed ) ; fci absent ( module of not completed for any item ) ; fci positive ( compiled for each item ) . in the end we analysed which information was lacking in the fci negative class the different guidelines proposed in the european union , despite being similar in many parts , have undergone adaptations depending on the epidemiological situation of each state . all these adaptations have occurred as the results of inspections carried out by the central and/or the food veterinary office ( fvo ) . in particular , these adaptations took into consideration the diseases occurring in each country . the forms varied from very detailed on specific diseases to forms in which only symptoms are requested ( affsa , 2008 ) . considering the animal specificity of the models , the majority of the examined countries only spain and germany have a single model regarding all species ( spanish government , 2009 ; german republic , 2004 ) . in general , the fci model of the poultry industry is the most detailed in all countries . most of the countries have paper forms ; but holland , uses computerised systems for small ruminants , for example . the fci model is sent the day before slaughtering with few exceptions . in france fci for poultry and rabbits is sent 48 h before slaughtering . in holland for sheep , goats and cattle it is possible to send fci simultaneously with the animals if few animals are sent for the slaughtering . a common finding is the fact that the fci models are collected by simple questions with direct answer ( yes / no ) and in the forms blanks for any additional information are provided . this situation results in large discrepancies such as : undifferentiated modules or modules differentiated by species , modules integrated or non - integrated to other mandatory documents ( e.g. mod . iv ) , modules with space or without space for additional data ( symptoms , pharmaceutical treatments ) . in general , almost all of the piedmont slaughterhouses considered do not require the document 24 h before the arrival of the animals . for all the species considered , the number of fci forms classified as negative is the most representative group ( from 78.89 to 99.1% ) and the models classified as positive are the smallest group ( from 0 to 7.14% ) . an important consideration must be ruled out related to the slaughtering capacity : the less the number of slaughtered animals , the more attention is paid by the veterinary service to check the forms . in addition to these general considerations our analysis highlighted some critical issues related to individual species . for cattle in the category fci negative , the missing data concerns , mainly , forms where pathologies were signaled but symptoms were not reported . in particular , those animals were sent for an emergency slaughtering or were accompanied by health certificates compiled by the private veterinarian . for the category fci absent the high percentage ( 8.25% ) recorded in a slaughterhouse was due to agreements between farmers and the fbo as a part of the supplier s accreditation process . it is clear that this is not just an arbitrary interpretation of the concept of the fci , but also improper extension of the accreditation process . another finding for cattle is that the fci is often considered data related to the farm and not the animals sent to the slaughterhouse . for this reason sometimes cattle from the same farm had more models n. iv and only one fci model . considering pigs , a peculiarity is due to the fact that some slaughterhouses export to australia , russian federation and china . for this slaughter plants , the information contained in the fci module gives additional health information being a mandatory request of the importing nation . considering the equine sector , a critical common point is the fact that many documents are written in the language of the country of origin only . as for small ruminants furthermore , the high percentage of parasitosis often found in post - mortem are not linked to an appropriate record neither of treatments nor of symptoms on the fci form . for poultry the possibility to perform ante - mortem inspection in the holding of provenance makes compulsory to fill out a health certificate in addition to the accompanying document . only in poultry slaughterhouses the fci form this agreement with the veterinary service enables the fbo to avoid the compilation of the health certificate representing a charge for him . in rabbits slaughtering the form was sent 24 h before the arrival of the animals due to the higher percentage of drug treatments . in the end , our analisys stressed out a certain number of criticities common for all the species considered . the fci negative is due mostly to the lack of information about symptoms even in the presence of health certificates and drug treatments . sometimes drug treatments are completely omitted on fci modules and on model n. iv or sometimes declared on the model n. iv and absent on fci modules . the information of livestock is almost always reported in reference to compulsory eradication plans for domestic cattle , pigs and small ruminants , while data are never reported to voluntary schemes or specific accreditation programmes . finally , we infer how in our country the real benefit derived from fci in terms of better health quality of food as a consequence of the uniformity and timely completion of this model is not yet fully understood ( franchini , 2004 ) . however , these findings are completely in line with what has been shown by the audit of the fvo in other european countries , such as ireland , lithuania and cyprus ( european commission , 2011 , 2012a , 2012b ) . given the large differences emerged , in our opinion it is essential to standardise the system by the aid of training courses and of new official fci forms , with guidelines for the compilation ( appendix ) . the eu reiterates the importance of increasing the level of food safety through the collection and transmission of information along the entire food chain . fci is a reliable tool to achieve it . from the data collected in this investigation the importance of fci seems not to be perceived by the fbo and also by the public veterinary service , which is often limited to assess the presence of the form , without checking its content . we believe that the issue of guidelines by the state - regions conference could in italy , like in other eu countries , give effectively increase the veterinary checks at slaughter and therefore favour the safety of the meat . abdominal pregnancies ( both primary and secondary ) have a reported incidence of 1 in 10,000 . worldwide , the incidence of ectopic pregnancies is increasing , with significant contribution from assisted reproductive technologies ( art ) . however , the incidence of abdominal pregnancies have remained constant or reduced , and maternal mortality reduced from 20% to less than 5% in last 20 years due to early diagnosis and management . early diagnosis has enabled laparoscopic management in many of these cases . in this study , we report a case of primary abdominal pregnancy following intra - uterine insemination ( iui ) managed laparoscopically . y.p , a 31-year - old female , attended our opd on 22 august 2010 , with chief complaints of amenorrhea of 2 months , following an iui cycle . she had primary infertility of 3 years , and was undergoing iui for mild - to - moderate male factor coupled with mild pelvic endometriosis . this was her first iui cycle ( cc1 00 mg d2-d6 , hcg trigger , and dydrogestrone as luteal support ) . the undisturbed ectopic gestation sac seen on the anterior surface of the uterus and anterior leaf of broad ligament the ectopic pregnancy site which started to bleed , on manipulation of the uterus bleeding ectopic sac separating from peritoneal surface protruding gestational sac bleeding from the implantation site after gestational sac was sucked out implantation site after removal of ectopic sac and hemostasis her previous menstrual cycles were regular . on transvaginal sonography , the uterus was bulky , very thick regular echogenic endometrium , and no gestational sac in cavity [ figure 7 ] . both ovaries were normal with a corpus luteum in the left ovary [ figures 8 and 9 ] . there was a 1.6 cm mass with strongly echogenic rim , close to the uterus , in the left side suggestive of an ectopic pregnancy sac . serum b - hcg in the same day was 9470 miu / ml [ figures 10 and 11 ] . uterus with empty endometrial cavity transvaginal scan showing left ovary with a corpus luteum image of the normal right ovary transvaginal sonography showing ectopic gestational sac in the region of the left adnexa transvaginal sonography picture showing both the empty uterus and adjacent ectopic sac laparoscopy was offered to the patient , who however , desperately requested for the conservative approach ( she had undergone diagnostic hystero - laparoscopy 2 months back during her infertility workup , and also did not want to loose a fallopian tube ) . she was given one intramascular injection of methotrexate ( 50 mg ) on 22.08.2010 following routine blood tests . however , follow - up of serial b - hcg on 26.08.2010 showed persisting levels of 9300 miu / ml and transvaginal sonography showed a size of the mass increased to 2 cm . was bulky , deviated to the right side with a few old and new endometriotic deposits on the antero - superior surface . the pregnancy sac was implanted on the peritoneal surface of the broad ligament , between the round ligament and the utero - vesical fold . initial appearance was a mound - like elevation , with yellowish brown margins [ figures 1 and 2 ] . there was significant bleeding from the base [ figure 5 ] , which was controlled with bipolar coagulation and superficial infiltration of pitressin [ figure 6 ] . -hcg fell to 203 iu within 4 days , and subsequently declined to non - pregnant levels . incidence has been reported as approximately 1 in 10,000 live births , and 9.2 per 1000 ectopic pregnancies . most of these are secondary abdominal pregnancies , which means the embryo had primarily implanted in the fallopian tube , extruded , or expelled and then secondarily implanted itself on another intra - abdominal surface . in primary abdominal pregnancy , which is the rarest type of ectopic gestation , the conceptus implants on the peritoneal surface . studdiford 's criteria used to diagnose primary abdominal pregnancy are described as : the presence of normal bilateral tubes and ovaries with no evidence of recent or past pregnancy.no evidence of a uteroperitoneal fistula.the presence of pregnancy related exclusively to the peritoneal surface , early enough to eliminate the possibility of secondary implantation after primary tubal nidation . the presence of normal bilateral tubes and ovaries with no evidence of recent or past pregnancy . the presence of pregnancy related exclusively to the peritoneal surface , early enough to eliminate the possibility of secondary implantation after primary tubal nidation . alternatively , a classification relevant from diagnosis and treatment defines early peritoneal pregnancy ( 20 weeks of gestation ) or advanced ( 20 weeks of gestation ) . primary abdominal pregnancies have been reported from omentum , sigmoid colon , posterior peritoneum of pelvis , spleen , liver , diaphragm , obturator foramen , posterior surface of uterus , retroperitoneum , and pancreas.[721 ] primary peritoneal pregnancy has been reported in relation to foci of endometriosis . rare and new varieties of ectopics such as heterotopic pregnancies , cervical , cesarean scar pregnancies have surfaced . however , the incidence of abdominal pregnancies has not changed , may be reduced due to early diagnosis . presentation of patents with primary abdominal pregnancy varies greatly , however , with early diagnosis like our case , patients may be totally asymptomatic . in earlier literature abdominal pregnancies were typically diagnosed late . now with serial b - hcg , excellent ultrasonography , and mri facilities , these pregnancies are diagnosed much earlier . our case is unique in some aspects . to our knowledge , this is the only case reported of primary peritoneal pregnancy in the anterior peritoneal leaf of the broad ligament following intrauterine insemination . the presence of uterine surface endometriosis was documented by a routine diagnosis laparoscopy only 2 months earlier . prognosis for future fertility also appears good following early diagnosis , as the tubes , ovaries are not directly involved in pregnancy , nor removed during the operative procedure and are not involved in postoperative adhesion formation .

food chain information ( fci ) is an innovation of the new european regulation . its purpose is to enhance the concept of food security . fci includes specifications such as : health status , information on treatments and diseases , analytical reports on control plans , zoonoses or environmental contaminants , production performance , etc . the aim of this article is to compare the different european guidelines and analyse the situation in piedmont in order to assess potential problems and propose solutions . european guidelines are similar one another , but they have been tailored to the epidemiological situations of each state . except for spain and germany , fci models are different for each species and the poultry sector is the most detailed . unfortunately , italy has not provided guidelines yet , and this has generated considerable differences . overall , the number of fci models with incomplete information is the largest group compared to the models not completed for each entry . the main deficiencies are related to pharmacological treatments . the health status of the farm is listed consistently regarding the compulsory eradication plans , but other national voluntary or accreditation plans are rarely mentioned . the situation is similar in other european countries . in conclusion , fci is an effective tool if applied with consistency and reason . only in this way the collection of data will be effective and representative of the food chain . primary abdominal pregnancy is an extremely rare type of extrauterine pregnancy . it has been reported from many unusual intra - abdominal sites . we report a case of primary abdominal pregnancy following intra - uterine insemination ( not reported earlier to our knowledge ) . implanted on the anterior surface of the uterus possibly related to an endometriotic foci . early diagnosis enabled laparoscopic management of this case .

nursing science as well as mental health care in general has , in fact , comprised both the humanistic paradigm that can be seen as a bottom - up approach , where the patient 's individual experiences are considered , and the biomedical model that can be regarded as a top - down approach toward the patient , where the professionals are viewed as the experts ( lindsey & hartrick , 1996 ) . the organizational processes such as deinstitutionalization and integration of patients in community - based mental health services have made the challenge to create a clear definition of health at all levels in mental health care even more essential ( magnusson , hgberg , ltzen , & severinsson , 2004 ) . an important distinction has been made between illness prevention , which can be described as the avoidance of disease , and health promotion that comprises a number of activities seeking to expand positive potentials for health ( pender , 1996 ) . it has been maintained that , although a more holistic health perspective has been developed in nursing science in recent decades , nursing research sometimes still tends to characterize health as the absence of symptoms of disease or handicap ( hwu , coates , & boore , 2001 ) . a divergence has , however , been recognized in the nursing discipline between these two contrasting health care paradigms ( malin & teasdale , 1991 ) . a focus in health care that remains mainly on illness and deficits tends to reinforce the patient 's experience of illness and disability ( simmons , 1989 ; svedberg , jormfeldt , & arvidsson , 2003 ) . however , the main focus in previous research regarding health in mental health care has been to alleviate and reduce mental illness and symptoms of disease , while frequently overlooking the patients needs of support for their health from a subjective perspective ( berg & sarvimki , 2003 ) . psychiatric symptoms are , from the perspective of pathogenesis , viewed as signs of a malfunctioning in the brain and the main goal of health care is to cure or prevent illness . from this perspective the concept of health is viewed as normality and disease is viewed as a deviation from this normality . according to this perspective , interventions such as prescribing a pill aiming at alleviating illness and symptoms of disease are often simultaneously viewed as health promoting as health and illness are seen as endpoints of the same continuum . however , health can not sufficiently be explained in a negative way as absence of symptoms because such an explanation tends to neglect the positive aspects of health ( downie et al . , 1996 ; hedelin & strandmark , 2001 ; naidoo & wills , 2000 ) . thus it is of great importance that the staff in mental health care develops a willingness to respect the dignity of patients and to become aware of the patients possibilities and resources ( jormfeldt , svedberg , fridlund , & arvidsson , 2007 ; svedberg et al . , 2003 ) . the adoption of a positive attitude and focus on possibilities has been proposed to motivate people with long - term mental health problems to help them engage in and to take an active part in their own care ( repper , ford , & cooke , 1994 ) . the perspective of salotogenesis is built on knowledge about health , how it is achieved , and incorporates a continuous view of health and illness . the individual is viewed as a whole person in relation to his or her environment and the main goal from this perspective is to strengthen and promote health . from a holistic perspective health care of today seems to take for granted that interventions aiming at alleviating symptoms of disease automatically promote processes of health as well . however , clinical experiences from mental health nursing show that this is not necessarily the case . perhaps the promotion of health processes reduces the process of illness to a greater extent than the prevailing , mainly biomedical , clinical praxis of today is able to promote those health processes . as an example from the biomedical perspective , the diagnosis of schizophrenia is often described as incurable and the patient is often instructed to follow doctors prescriptions very carefully because medication is seen as the foundation for treatment and the most superior intervention to alleviate the symptoms ( van os et al . , 2006 ) . this intervention may place our patient in the position where the symptoms are partly alleviated but the patient is still at great risk of becoming ill in the event of stressful situations . it is easy to understand that the patient 's motivation to maintain hope and follow prescriptions can sometimes be reduced if his or her problems are primarily viewed from this perspective . from a health perspective , the goals and desires of the individual are vital and serve as guidance for the care . the process of reaching individual goals is seen as the superior pathway for a cure from mental illness . an intervention to support our imagined patient to reach his individual goals may place him / her in a position where he / her has a good chance of achieving a good life and a feeling of well - being . if or when medication is used , from this perspective , it is to support the patient in his / her process of reaching his / her goals from a salutogenic perspective of health . valid and reliable measures of a positive multidimensional concept of health in mental health services need to address autonomy , social involvement , and comprehensibility ( jormfeldt , svensson , arvidsson , & hansson , 2008 ) . autonomy incorporates the ability to function in daily life , self - esteem , and to feel positively about oneself . perceptions among nurses and patients in mental health services have been shown to be very much alike ( jormfeldt , 2010 ; jormfeldt , svedberg , & arvidsson , 2003 ; svedberg , jormfeldt , fridlund , & arvidsson , 2004 ) . however , one difference between nurses and patients expressions of health is the importance of feelings of being equal and important to other people . these feelings were related to a feeling of self - worth and health , which nurses working in mental health services do not always express an awareness of ( jormfeldt et al . , 2003 ) . patients in mental health services have described the importance of being looked upon just as anyone else as well as the need for help to reduce the shame of having to request and receive psychiatric care in order to reduce stigmatization and enhance health ( schrder , ahlstrm , & larsson , 2006 ) . ( 2002 ) found that overall self - esteem was predicted by being satisfied with family relationships and that having at least one close friend was a predictor of positive self - esteem , while being able to cope without friends was associated with superior self - esteem . self - esteem has shown to be strongly associated with health while psychiatric symptoms to a much lesser extent have shown to be negatively associated to health ( jormfeldt , arvidsson , svensson , & hansson , 2008 ) . social involvement contains experiences of attachment to others and feelings of being both a recipient and a donor of social support . mental health patients emphasize the importance of experiences of participation in social contexts and being able to give support to others , while the nurses more often speak of the importance of patients experiencing trust and being part of a social context ( jormfeldt et al . , 2007 ) . hedelin and jonsson ( 2003 ) describe mutuality in relationships between people as a major element in their experience of mental health . the essence of mental health has been defined as the experience of confirmation by means of being noticed , respected , and regarded as a valuable person by others ( hedelin & strandmark , 2001 ) , and one very important aspect of health is to feel included in a social network . ( 1999 ) have studied clinical characteristics that may be related to quality of life without finding any associations with global subjective quality of life , while one objective indicator , i.e. , to have at least one close friend , was associated with superior self - rated quality of life . unmet needs in the domain of social relationships are related to a worse quality of life ( hansson et al . , 2003 ) . involuntary loneliness and the experience of not belonging to anyone have been described as a lack of participation in the world ( dahlberg , 2007 ) . comprehensibility includes the understanding and awareness of one 's own situation , how the situation has emerged , and a view of how to get the situation changed if desired . comprehensibility could be recognized as meaningfulness , being at peace , and looking forward to something positive , as well as personal growth and seeing suffering as a motivating force to change ( jormfeldt et al . , 2003 ) . health as a process includes the view of suffering as a motivator for development toward change and increased awareness ( jormfeldt et al . , 2007 ) as described by several previous researchers ( herberts & eriksson , 1995 ; lindsey , 1996 ; long , 1998 ) . jones and meleis ( 1993 ) maintained that increased self - esteem , which is often enhanced through empowerment processes , is essential in order to gain the energy that is needed to be able to fully use one 's own personal health capacity . a number of earlier researchers have depicted health as an increased awareness and a foundation for increased well - being ( eriksson , 1984 ; long , 1998 ; moch , 1998 ; parse , 1990 ) . the process toward increased awareness and greater personal meaning has been labeled transition and could be encouraged through communication of thoughts and needs ( skrster & willman , 2006 ) . this awareness can , in turn , lead to more constructive behavior in accordance with individual wishes related to the environment . positive mental health has to be promoted at the individual level ( mezzich , 2005 ) and include factors such as acceptance , faith , hope , meaningfulness , and meaningful relationships ( edward , welch , & chater , 2009 ) . according to this reasoning , the concept of health is far beyond the limits of the wellness illness continuum ( hwu , coates , boore , & bunting , 2002 ; moch , 1998 ) . health promotion interventions are determined by the definition of health . to be able to truly promote health in mental health care , we have to define health in a positive manner and not only as an absence of illness or disease . the construct of remission can be seen as an example of measuring absence of symptoms of illness as a measure of health . the symptomatic remission criteria among patients diagnosed with bipolar disorder or schizophrenia is used as an assessment of reduction of symptoms . the underlying presumption is that remission is a prerequisite for everyday functioning among these patients . remission is suggested to be a good measure of treatment outcome and it is also proposed that remission of clinical symptoms is a required first step toward functional recovery ( van os et al . , 2006 ) . on the other hand , the construct of remission pays little attention to the subjective experience of the patient and the focus is entirely on the absence of symptoms of illness even though the concept of remission often is described as a positive concept related to recovery , health , and quality of life . the difficulties in measuring subjectively experienced health in individuals or in populations as well as in mental health services without a clear definition of the positive dimension of health has been highlighted by jormfeldt et al . the overall goal of nursing in general health care as well as in mental health care is to promote the patient 's subjective experience of health . an important measure of quality of care is thus to assess to what extent this goal is reached . the more ambiguous the definition of health is , the less importance the concept will be given and other more clarified concepts , as symptoms of illness or disease , will be given greater significance when mental health care is evaluated . in health care it has traditionally been easier and more common to measure absence of symptoms of disease as these symptoms often are more clearly defined then subjectively experienced health . studies evaluating questionnaires measuring patients subjectively experienced health in mental health services are rare , and most questionnaires used to measure health in health care have , in fact , been measuring absence of illness in terms of symptoms and disabilities in line with the biomedical view of health ( christiansen & kooiker , 1999 ; whitehead , 2003 ) . consequently it is often the negative dimension of the concept of health that is being measured , evaluated , and used as a base for evidence . most evidence - based models appear to understate the importance of non - pharmacological interventions and a lack of knowledge about such interventions is recognized ( hayman - white & happel , 2007 ) . nursing care and caring science requires humanistic knowledge with a hermeneutical dimension to achieve real progress in caring ( eriksson , 2002 ) , the challenge is to shape an evidence base for this kind of knowledge . health is to a great extent associated with self - esteem and to a lesser extent with clinical characteristics and psychiatric symptoms . mental health care must consequently involve interventions , relationships , and contexts primarily aiming to support the patient 's self - esteem and empowerment if it is to promote health . the key to enhanced health among patients in mental health services is to acquire support regarding self - esteem , empowerment , and quality of life . to view the goal of mental health care mainly as reducing symptoms should not be sufficient when mental health as well as overall health is to such a low level explained purely by psychiatric symptoms . subjectively experienced health is one of the most important outcome measures of the quality of care provided to patients in mental health care irrespective of type of symptom , illness , or disability . advanced analysis of the concept of health may contribute to enhanced empowerment strategies in health care . further research of importance would be to investigate the effects and effectiveness of empowerment approaches in mental health services and to consider every possible causal relationship between psychiatric symptoms and health in order to develop and implement more constructive intervention approaches in mental health services . clinical interventions aiming at strengthening positive dimensions of health and self - esteem are required in mental health care in order to meet the patients needs of enhanced health and to make further investigations possible regarding effects of such interventions . the author has not received any funding or benefits from industry or elsewhere to conduct this study . glycemic abnormalities and diabetes are on the rise globally ( 1 ) . according to the most recent statistics , 9.3% of the u.s . population , 29.1 million individuals , live with diabetes , and the level of glycemia in the general public ( mean fasting plasma glucose ) since 1980 has risen by 2.5 mg / dl per decade in women , and by 3.2 mg / dl per decade in men ( 1,2 ) . hyperglycemia is common after stressful events , such as myocardial infarction , stroke , and sepsis , or in the postoperative setting , after cardiac surgery ( 3 ) . stress - induced hyperglycemia is a transient phenomenon , distinct from the chronic glucose dysregulation brought about by diabetes ( 3 ) . studies ( 47 ) have shown that stress hyperglycemia after cardiac surgery , which occurs in patients both with and without diabetes , is associated with a higher risk of complications , including major infections , and increased mortality . the management of stress hyperglycemia in patients receiving critical care is a matter of great controversy ( 8) . the rationale of glucose control management rests on the hypothesis that the relationship between hyperglycemia and adverse outcomes is one of causation . trials assessing the potential benefits of strict glycemic control ( target range 80110 mg / dl ) ( 912 ) have produced conflicting results , with early studies reporting decreased mortality and morbidity , and subsequent studies showing a lack of benefits or even worse outcomes , along with an increased risk of hypoglycemia . these trials included a heterogeneous selection of patients , which may have influenced the response to short - term changes in glucose levels . given the uncertainty about the effectiveness of different protocols targeting normoglycemia , most medical societies have endorsed a moderate approach to glucose control in perioperative and critical care settings , recommending that patients , regardless of their diabetes status , have their serum glucose levels maintained at < 180 mg / dl ( 6,13 ) . more recently , due the ongoing debate , the surgical care improvement project , a national program undertaken to improve outcomes in surgery whose measures are publicly reported on the centers for medicare and medicaid hospital website and affect reimbursement , has suspended its recommendation on maintaining postoperative glucose levels at < 180 mg / dl ( 14 ) . an increasing body of evidence shows that the association between stress hyperglycemia and adverse outcomes varies depending on the pre - existence of diabetes ( 3,1517 ) . although diabetes is a heterogeneous disease with a broad spectrum of manifestations and symptom severity ( 18 ) , most of the previous studies have analyzed the impact of stress hyperglycemia in diabetes without further stratification by prior treatment . however , prior treatment history and degree of glycemic control may be important effect modifiers ( 19 ) . consideration of these factors would permit the selection of more appropriate glucose targets for specific groups of patients , particularly in intensive care , where complications can be life threatening and costs are the highest . the purpose of this study is to assess the clinical and economic outcomes associated with postoperative hyperglycemia among patients without and with diabetes with different treatment histories who have undergone cardiac surgery . between february and october 2010 , the cardiothoracic surgical trials network conducted a multicenter prospective cohort study to assess the incidence of hospital - acquired infections . all adult cardiac surgery patients ( 18 years old ) without pre - existing infection on hospital admission were eligible to participate ( n = 5,158 ) ( 20 ) . of the 10 participating centers ( 9 american and 1 canadian ) , only patients from u.s . centers ( n = 4,614 ) were included in order to avoid the confusion of mixing data from different health care systems with very different reimbursement methods . billing data for these nine centers were obtained from the university healthsystem consortium , an alliance of u.s . academic medical centers with the goal of promoting improvements in the quality , safety , and efficiency of health care . the final study population included 4,316 patients in whom glucose was measured within 48 h after surgery . the study protocol was approved by the institutional review boards of each of the participating study centers . baseline variables prior to surgery included the following : demographics , anthropometrics , laboratory results , and comorbid conditions . pre - hospital admission diabetes status was defined by prior therapy with oral antidiabetes medication only , a history of non insulin - treated diabetes mellitus ( nitdm ) , or a history of insulin - treated diabetes mellitus ( itdm ) . the latter group included patients treated with insulin only or a combination of insulin and oral antidiabetic medications . ( hba1c ) was assessed preoperatively in patients with diabetes . in 83 nitdm patients ( 15% ) and 66 itdm patients ( 16% ) glomerular filtration rate ( gfr ) was estimated from serum creatinine using the chronic kidney disease epidemiology collaboration equation ( 22 ) . surgical parameters included sternotomy , hospital admission type ( elective , urgent , emergent ) , procedure type ( isolated valve , isolated coronary artery bypass graft [ cabg ] , transplantation or ventricular assist device , cabg with valve , thoracic aortic , other ) , and surgery duration . the study protocol included blood glucose measurements every 6 h for 48 h after surgery . based on previous research using maximum blood glucose level as a measure of blood glucose control ( 23 ) , we used the highest value among these measurements . hyperglycemia was defined as having at least one measurement > 180 mg / dl . to convert glucose values to millimoles per liter , multiply by 0.0555 . participating centers provided their protocol for managing glucose in the intensive care unit ( icu ) after cardiac surgery . the standard protocol of each icu was then used to approximate the actual treatment that the individual patients of that hospital received . among the centers , three had guidelines recommending a target range for blood glucose concentration between 80 and 120 mg / ml , whereas the other six centers had a target range between 140 and 180 mg / ml . the protocols were grouped into the following two categories for the analysis : tight outcomes related to resource use included hospitalization costs and hospital length of stay ( los ) . clinical outcomes during the hospital stay were defined as death , major infection , cardiac complications , and respiratory complications . this method of approximating cost is widely used and provides reasonably accurate estimates of actual costs ( 24 ) . major infections were classified using definitions adapted from the centers for disease control and prevention / national healthcare safety network ( 20 ) . complications other than infections were identified through icd-9 codes , and were defined as cardiac complications ( code 9971 ) and respiratory failure ( codes 5185 , 51881 , 51882 , 51884 , and 7991 ) , with exclusion of those flagged as present on admission . previous studies related to the existence of possible differences between patients with and without diabetes , with respect to outcomes associated with stress hyperglycemia , were searched on pubmed using the terms critical care , critically ill , icu , hyperglycemia , and diabetes mellitus , and english language and human species were used as filters . the search retrieved 653 citations , including 254 review articles . after reviewing titles and abstracts , we divided the study population in subgroups of patients with no diabetes , nitdm , and itdm . differences in baseline characteristics were assessed using kruskal - wallis one - way anova for continuous variables and tests for categorical variables . the relationship between maximum glucose levels and outcomes models included all baseline variables and interactions between a variable indicating diabetes status ( no diabetes , nitdm , and itdm ) and maximum glucose levels when analyzed continuously , or hyperglycemia when analyzed dichotomously . predictors were selected using costs as outcome and the akaike information criterion , which calculates the log - likelihood penalized for the number of parameters included . final regression models included the maximum glucose - diabetes treatment interaction term , age , sex , race ( white vs. other ) , bmi , white blood cell count , gfr , hemoglobin level , ejection fraction , renal insufficiency , lung disease , congestive heart failure , prior cardiac surgery , corticosteroid use , hospital admission type ( elective / urgent / emergent ) , procedure type , surgery duration , thoracic approach , and medical center . within an analysis restricted to patients with diabetes missing hba1c values were imputed by multiple imputation with a multivariable algorithm including all predictors , costs , hospital los , and the composite end point of complications . nonlinear associations of continuous variables were modeled by restricted cubic spline functions with four knots ( 25 ) . for estimating costs , we used a distribution with a log link function ; for hospital los , we used a negative binomial distribution with a log link function ; and for complications , we used logistic regression . to demonstrate the adjusted change in each outcome with varying glucose levels , we predicted the outcome of interest in each patient by using the regression equation and varying blood glucose concentrations ( between 120 and 300 mg / dl when continuous , and > 180 vs. 180 mg / dl when dichotomous ) while keeping the other parameters fixed at their observed value . to take into account parameter uncertainty , regression models were refitted in 1,000 bootstrap data sets , and subsequently predictions were made in each bootstrap . after ordering the 1,000 bootstrap estimates , 2.5% and 97.5% quantiles were used for calculating 95% cis . to examine how a routine implementation of the recommended threshold < 180 mg / dl would affect our different patient subgroups , we analyzed the expected outcomes above and below this threshold . differences in the individual complications were adjusted for age , sex , and type of procedure only . each scenario represented the implementation of a different glucose control threshold ranging from 120 to 300 mg / dl , and assumed the perfect situation in which patients glucose levels were maintained below this threshold . per scenario , patients were reassigned a maximum glucose value equal to the threshold when their actual value was higher . at lower thresholds , more patients have a glucose value above the threshold and will be reassigned to the threshold value ; whereas , at higher thresholds , more patients will keep their actual glucose value . finally , we graphically depicted the difference between the expected outcome of each scenario and the originally observed outcome . this difference will tend to zero at higher thresholds , given that more patients will keep their actual glucose levels . all analyses were performed using r version 3.1.0 ( r foundation for statistical computing [ http://www.r-project.org/ ] ) . between february and october 2010 , the cardiothoracic surgical trials network conducted a multicenter prospective cohort study to assess the incidence of hospital - acquired infections . all adult cardiac surgery patients ( 18 years old ) without pre - existing infection on hospital admission were eligible to participate ( n = 5,158 ) ( 20 ) . of the 10 participating centers ( 9 american and 1 canadian ) , only patients from u.s . centers ( n = 4,614 ) were included in order to avoid the confusion of mixing data from different health care systems with very different reimbursement methods . billing data for these nine centers were obtained from the university healthsystem consortium , an alliance of u.s . academic medical centers with the goal of promoting improvements in the quality , safety , and efficiency of health care . the final study population included 4,316 patients in whom glucose was measured within 48 h after surgery . the study protocol was approved by the institutional review boards of each of the participating study centers . baseline variables prior to surgery included the following : demographics , anthropometrics , laboratory results , and comorbid conditions . pre - hospital admission diabetes status was defined by prior therapy with oral antidiabetes medication only , a history of non insulin - treated diabetes mellitus ( nitdm ) , or a history of insulin - treated diabetes mellitus ( itdm ) . the latter group included patients treated with insulin only or a combination of insulin and oral antidiabetic medications . ( hba1c ) was assessed preoperatively in patients with diabetes . in 83 nitdm patients ( 15% ) and 66 itdm patients ( 16% ) glomerular filtration rate ( gfr ) was estimated from serum creatinine using the chronic kidney disease epidemiology collaboration equation ( 22 ) . surgical parameters included sternotomy , hospital admission type ( elective , urgent , emergent ) , procedure type ( isolated valve , isolated coronary artery bypass graft [ cabg ] , transplantation or ventricular assist device , cabg with valve , thoracic aortic , other ) , and surgery duration . the study protocol included blood glucose measurements every 6 h for 48 h after surgery . based on previous research using maximum blood glucose level as a measure of blood glucose control ( 23 ) , we used the highest value among these measurements . hyperglycemia was defined as having at least one measurement > 180 mg / dl . to convert glucose values to millimoles per liter , participating centers provided their protocol for managing glucose in the intensive care unit ( icu ) after cardiac surgery . the standard protocol of each icu was then used to approximate the actual treatment that the individual patients of that hospital received . among the centers , three had guidelines recommending a target range for blood glucose concentration between 80 and 120 mg / ml , whereas the other six centers had a target range between 140 and 180 mg / ml . the protocols were grouped into the following two categories for the analysis : tight outcomes related to resource use included hospitalization costs and hospital length of stay ( los ) . clinical outcomes during the hospital stay were defined as death , major infection , cardiac complications , and respiratory complications . hospitalization costs were calculated from the billing data using medicare cost center specific cost - to - charge ratios . this method of approximating cost is widely used and provides reasonably accurate estimates of actual costs ( 24 ) . major infections were classified using definitions adapted from the centers for disease control and prevention / national healthcare safety network ( 20 ) . complications other than infections were identified through icd-9 codes , and were defined as cardiac complications ( code 9971 ) and respiratory failure ( codes 5185 , 51881 , 51882 , 51884 , and 7991 ) , with exclusion of those flagged as present on admission . previous studies related to the existence of possible differences between patients with and without diabetes , with respect to outcomes associated with stress hyperglycemia , were searched on pubmed using the terms critical care , critically ill , icu , hyperglycemia , and diabetes mellitus , and english language and human species were used as filters . the search retrieved 653 citations , including 254 review articles . after reviewing titles and abstracts , we divided the study population in subgroups of patients with no diabetes , nitdm , and itdm . differences in baseline characteristics were assessed using kruskal - wallis one - way anova for continuous variables and tests for categorical variables . the relationship between maximum glucose levels and outcomes models included all baseline variables and interactions between a variable indicating diabetes status ( no diabetes , nitdm , and itdm ) and maximum glucose levels when analyzed continuously , or hyperglycemia when analyzed dichotomously . predictors were selected using costs as outcome and the akaike information criterion , which calculates the log - likelihood penalized for the number of parameters included . final regression models included the maximum glucose - diabetes treatment interaction term , age , sex , race ( white vs. other ) , bmi , white blood cell count , gfr , hemoglobin level , ejection fraction , renal insufficiency , lung disease , congestive heart failure , prior cardiac surgery , corticosteroid use , hospital admission type ( elective / urgent / emergent ) , procedure type , surgery duration , thoracic approach , and medical center . within an analysis restricted to patients with diabetes missing hba1c values were imputed by multiple imputation with a multivariable algorithm including all predictors , costs , hospital los , and the composite end point of complications . nonlinear associations of continuous variables were modeled by restricted cubic spline functions with four knots ( 25 ) . for estimating costs , we used a distribution with a log link function ; for hospital los , we used a negative binomial distribution with a log link function ; and for complications , we used logistic regression . to demonstrate the adjusted change in each outcome with varying glucose levels , we used the recycled prediction method ( 26 ) . in brief , we predicted the outcome of interest in each patient by using the regression equation and varying blood glucose concentrations ( between 120 and 300 mg / dl when continuous , and > 180 vs. 180 mg / dl when dichotomous ) while keeping the other parameters fixed at their observed value . to take into account parameter uncertainty , regression models were refitted in 1,000 bootstrap data sets , and subsequently predictions were made in each bootstrap . after ordering the 1,000 bootstrap estimates , how a routine implementation of the recommended threshold < 180 mg / dl would affect our different patient subgroups , we analyzed the expected outcomes above and below this threshold . differences in the individual complications were adjusted for age , sex , and type of procedure only . each scenario represented the implementation of a different glucose control threshold ranging from 120 to 300 mg / dl , and assumed the perfect situation in which patients glucose levels were maintained below this threshold . per scenario , patients were reassigned a maximum glucose value equal to the threshold when their actual value was higher . at lower thresholds , more patients have a glucose value above the threshold and will be reassigned to the threshold value ; whereas , at higher thresholds , more patients will keep their actual glucose value . finally , we graphically depicted the difference between the expected outcome of each scenario and the originally observed outcome . this difference will tend to zero at higher thresholds , given that more patients will keep their actual glucose levels . all analyses were performed using r version 3.1.0 ( r foundation for statistical computing [ http://www.r-project.org/ ] ) . the average age in the study population was 65.5 years , and 66% were male . among patients with diabetes , 43% were itdm patients . these patients generally had glucose levels that were less well controlled than nitdm patients , as indicated by their hba1c levels . moreover , patients with itdm had lower renal function , and a higher prevalence of congestive heart failure and chronic lung disease compared with the other patient groups ( table 1 ) . hyperglycemia during the first 48 h after surgery was reported in 70% of patients with diabetes and 36% of patients without diabetes . hyperglycemia was less frequent in hospitals with a tight glucose control protocol compared with hospitals with nontight glucose control . hypoglycemia , conversely , was more frequent in hospitals with tight glucose control , particularly in itdm patients ( table 1 ) . these results were confirmed in multivariable analyses , where the presence of a tight glucose control standard protocol in the hospital icu increased the likelihood of the development of hypoglycemia in itdm patients compared with a nontight protocol ( odds ratio of hypoglycemia in itdm patients = 2.39 [ 95% ci 1.64 to 3.48 ] ) . patients baseline characteristics continuous variables are reported as the median ( interquartile range ) , and categorical variables as n ( proportion ) . the test was performed for comparing categorical variables , and kruskal - wallis one - way anova was performed for comparing continuous variables . dm , diabetes mellitus ; na , not applicable ; vad , ventricular assist device implantation or explantation ; wbc , white blood cell . * the standard protocol of the icu had a blood glucose target range within 140180 mg / dl ( n = 2,816 ) . * * the standard protocol of the icu had a blood glucose target range within 80120 mg / dl ( n = 1,500 ) . itdm patients had , on average , higher cost and longer hospital los than patients with no diabetes or nitdm . there were 29 ( 0.9% ) , 5 ( 0.9% ) , and 7 ( 1.7% ) in - hospital deaths , respectively , among patients with no diabetes , nitdm , and itdm . furthermore , there were remarkable differences in the relationship of increasing glucose levels with resource use and clinical outcomes among patients with no diabetes , nitdm , and itdm ( fig . 1 ) . in patients without diabetes , the cost of hospitalization , hospital los , and the risk of complications increased with increasing glucose levels . in nitdm patients , cost , hospital los , and risk of complications peaked at 220 mg / dl , and slightly decreased with further glycemic increases . in contrast , in itdm patients , cost , hospital los , and the risk of complications during the hospital stay were highest at glucose concentrations < 180 mg / dl , and decreased with increasing glucose values , reaching a minimum between 180 and 240 mg / dl . persistent hyperglycemia , which was defined as two or more measurements > 180 mg / ml , occurred in 12.3% of patients without diabetes and 45.9% of patients with diabetes . in itdm patients , an increase in the number of hyperglycemic measurements was associated with reduced cost , number of complications , and hospital los , compared with patients with no hyperglycemia . conversely , in patients with no diabetes and patients with nitdm , such an increase correlated with higher cost , more complications , and prolonged hospital los ( supplementary fig . hba1c was significantly higher in itdm patients than in nitdm patients ( 7.5 vs. 6.7% [ 58 vs. 50 mmol / mol ] ) on average ( table 1 ) . in patients with diabetes , outcomes improved with increasing hba1c values . however , these improvements were not statistically significant in the multivariable complete case analysis . relative changes were 0.91 ( 95% ci 0.80 to 1.04 ) for the odds of complications , 0.98 ( 95% ci 0.95 to 1.00 ) for hospital los , and 0.98 ( 95% ci 0.95 to 1.00 ) for total costs . estimates based on multiple imputation were not different from those based on complete case analysis . among patients without diabetes , the cost of those with hyperglycemia was , on average , nearly $ 10,000 higher than the cost of those without hyperglycemia ; whereas , among itdm patients , the cost of those patients with hyperglycemia was on average approximately $ 15,000 lower ( table 2 ) . after adjustment for baseline and procedure variables , the change in cost with hyperglycemia was approximately an extra $ 3,000 in patients without diabetes and a decrease of $ 6,000 in patients with itdm . in patients with nitdm , the change in cost with hyperglycemia was positive but not significant . adjusted changes in hospital los associated with hyperglycemia followed a pattern similar to that of cost , with 0.8 additional days ( 95% ci 0.4 to 1.3 ) in patients without diabetes , and a decrease of 1.6 days ( 95% ci 3.7 to 0.4 ) in itdm patients , although the latter did not reach statistical significance . clinical and economic outcomes associated with postoperative hyperglycemia dm , diabetes mellitus . * adjusted for age , sex , race , bmi , white blood cell count , gfr , hemoglobin , history of heart failure , renal insufficiency , ejection fraction , prior cardiac surgery , history of lung disease , corticosteroids , surgery time , sternotomy performed yes / no , surgery type , procedure , and study site . * * adjusted for age , sex , and procedure . in patients without diabetes , after adjustment , hyperglycemia was associated with a 1.6% ( 95% ci 0.5 to 2.8 ) increased risk of major infections , a 2.6% ( 95% ci 0.0 to 5.3 ) increased risk of respiratory complications , and a trend toward increased cardiac complications ( table 2 ) . in nitdm patients , the risk of such complications associated with hyperglycemia increased as well , but without reaching statistical significance . conversely , in patients with itdm , hyperglycemia was associated with a reduced risk of adverse outcomes , particularly with respect to respiratory complications ( 12.5% [ 95% ci 22.4 to 3.0 ] ) and major infections ( 4.1% [ 95% ci 9.1 to 0.0 ] ) . in the analysis of hypothetical scenarios of different glucose thresholds , the outcomes after cardiac surgery predicted in patients without diabetes and patients with ntidm would improve with lowering postoperative glucose values . the expected benefits comprised an approximate $ 2,000 cost reduction and a 515% reduction in complications . in contrast , in patients with itdm , thresholds < 180 mg / dl would be harmful , although in this range outcomes were uncertain ( fig . the average age in the study population was 65.5 years , and 66% were male . among patients with diabetes , 43% were itdm patients . these patients generally had glucose levels that were less well controlled than nitdm patients , as indicated by their hba1c levels . moreover , patients with itdm had lower renal function , and a higher prevalence of congestive heart failure and chronic lung disease compared with the other patient groups ( table 1 ) . hyperglycemia during the first 48 h after surgery was reported in 70% of patients with diabetes and 36% of patients without diabetes . hyperglycemia was less frequent in hospitals with a tight glucose control protocol compared with hospitals with nontight glucose control . hypoglycemia , conversely , was more frequent in hospitals with tight glucose control , particularly in itdm patients ( table 1 ) . these results were confirmed in multivariable analyses , where the presence of a tight glucose control standard protocol in the hospital icu increased the likelihood of the development of hypoglycemia in itdm patients compared with a nontight protocol ( odds ratio of hypoglycemia in itdm patients = 2.39 [ 95% ci 1.64 to 3.48 ] ) . patients baseline characteristics continuous variables are reported as the median ( interquartile range ) , and categorical variables as n ( proportion ) . the test was performed for comparing categorical variables , and kruskal - wallis one - way anova was performed for comparing continuous variables . dm , diabetes mellitus ; na , not applicable ; vad , ventricular assist device implantation or explantation ; wbc , white blood cell . * the standard protocol of the icu had a blood glucose target range within 140180 mg / dl ( n = 2,816 ) . * * the standard protocol of the icu had a blood glucose target range within 80120 mg / dl ( n = 1,500 ) . itdm patients had , on average , higher cost and longer hospital los than patients with no diabetes or nitdm . there were 29 ( 0.9% ) , 5 ( 0.9% ) , and 7 ( 1.7% ) in - hospital deaths , respectively , among patients with no diabetes , nitdm , and itdm . furthermore , there were remarkable differences in the relationship of increasing glucose levels with resource use and clinical outcomes among patients with no diabetes , nitdm , and itdm ( fig . 1 ) . in patients without diabetes , the cost of hospitalization , hospital los , and the risk of complications increased with increasing glucose levels . in nitdm patients , cost , hospital los , and risk of complications peaked at 220 mg / dl , and slightly decreased with further glycemic increases . in contrast , in itdm patients , cost , hospital los , and the risk of complications during the hospital stay were highest at glucose concentrations < 180 mg / dl , and decreased with increasing glucose values , reaching a minimum between 180 and 240 mg / dl . persistent hyperglycemia , which was defined as two or more measurements > 180 mg / ml , occurred in 12.3% of patients without diabetes and 45.9% of patients with diabetes . in itdm patients , an increase in the number of hyperglycemic measurements was associated with reduced cost , number of complications , and hospital los , compared with patients with no hyperglycemia . conversely , in patients with no diabetes and patients with nitdm , such an increase correlated with higher cost , more complications , and prolonged hospital los ( supplementary fig . hba1c was significantly higher in itdm patients than in nitdm patients ( 7.5 vs. 6.7% [ 58 vs. 50 mmol / mol ] ) on average ( table 1 ) . in patients with diabetes , outcomes improved with increasing hba1c values . however , these improvements were not statistically significant in the multivariable complete case analysis . relative changes were 0.91 ( 95% ci 0.80 to 1.04 ) for the odds of complications , 0.98 ( 95% ci 0.95 to 1.00 ) for hospital los , and 0.98 ( 95% ci 0.95 to 1.00 ) for total costs . estimates based on multiple imputation were not different from those based on complete case analysis . among patients without diabetes , the cost of those with hyperglycemia was , on average , nearly $ 10,000 higher than the cost of those without hyperglycemia ; whereas , among itdm patients , the cost of those patients with hyperglycemia was on average approximately $ 15,000 lower ( table 2 ) . after adjustment for baseline and procedure variables , the change in cost with hyperglycemia was approximately an extra $ 3,000 in patients without diabetes and a decrease of $ 6,000 in patients with itdm . in patients with nitdm , adjusted changes in hospital los associated with hyperglycemia followed a pattern similar to that of cost , with 0.8 additional days ( 95% ci 0.4 to 1.3 ) in patients without diabetes , and a decrease of 1.6 days ( 95% ci 3.7 to 0.4 ) in itdm patients , although the latter did not reach statistical significance . clinical and economic outcomes associated with postoperative hyperglycemia dm , diabetes mellitus . * adjusted for age , sex , race , bmi , white blood cell count , gfr , hemoglobin , history of heart failure , renal insufficiency , ejection fraction , prior cardiac surgery , history of lung disease , corticosteroids , surgery time , sternotomy performed yes / no , surgery type , procedure , and study site . * * adjusted for age , sex , and procedure . in patients without diabetes , after adjustment , hyperglycemia was associated with a 1.6% ( 95% ci 0.5 to 2.8 ) increased risk of major infections , a 2.6% ( 95% ci 0.0 to 5.3 ) increased risk of respiratory complications , and a trend toward increased cardiac complications ( table 2 ) . in nitdm patients , the risk of such complications associated with hyperglycemia increased as well , but without reaching statistical significance . conversely , in patients with itdm , hyperglycemia was associated with a reduced risk of adverse outcomes , particularly with respect to respiratory complications ( 12.5% [ 95% ci 22.4 to 3.0 ] ) and major infections ( 4.1% [ 95% ci 9.1 to 0.0 ] ) . in the analysis of hypothetical scenarios of different glucose thresholds , the outcomes after cardiac surgery predicted in patients without diabetes and patients with ntidm would improve with lowering postoperative glucose values . the expected benefits comprised an approximate $ 2,000 cost reduction and a 515% reduction in complications . in contrast , in patients with itdm , thresholds < 180 mg / dl would be harmful , although in this range outcomes were uncertain ( fig . in this study , we demonstrated a significant association of postoperative stress hyperglycemia with economic and clinical outcomes , which varies with the presence of diabetes and its treatment . in patients with diabetes treated with insulin , glucose levels below the generally recommended threshold of 180 mg / dl conversely , in patients without and with diabetes not treated with insulin , adverse outcomes were decreased or unchanged . in these patient groups , the association between glucose levels and outcomes was characterized by a dose - response relationship with increasing complication rates at higher glucose levels . the results of this study suggest that targeting a glucose range below the threshold of 180 mg / dl , an approach in line with current guidelines , may be harmful in patients with diabetes treated with insulin . there are several possible explanations for a differential response among patients with and without itdm . first , metabolic homeostasis is altered in the cells of patients with diabetes , particularly in itdm patients ( 27 ) . the appropriate concentration of glucose for survival under stress - related conditions may be much higher in patients with itdm to provide energy for wound repair and to recover from major physiological insults ( 28 ) . the long - term exposure to higher blood glucose levels could result in the downregulation of glucose transporters limiting the influx of glucose across the cellular membrane ( 29 ) . independent of glucose level , higher hba1c levels decreased the likelihood of complications in patients with diabetes , as assessed with our composite end point . although it has been shown that higher hba1c values may decrease the risk of atrial fibrillation ( 30 ) and mortality in patients with acute hyperglycemia ( 31 ) , we found only a nonsignificant trend for decreasing the risk of complications . the protective effect mentioned above and a decreased risk of hypoglycemic events could be an explanation for this phenomenon . there is accumulating evidence that patients without a previous diagnosis of diabetes face a worse prognosis than patients with diabetes when stress hyperglycemia occurs ( 15,32 ) . in a large retrospective analysis ( 33 ) , the association between mortality risk and hyperglycemia in critically ill patients without diabetes was significantly stronger than in patients with diabetes . more recently , an increased risk of postoperative adverse events , such as myocardial infarction , stroke , transient ischemic attack , infectious complication , and renal insufficiency , was linked to hyperglycemia in patients without diabetes , but not in patients with diabetes ( 34 ) . in contrast with our findings , we found earlier reports ( 6,35,36 ) showing that lower glucose levels in patients with diabetes were linked to lower mortality and lower infection rates . however , the mode of insulin delivery in these earlier studies may not be directly comparable to the current glucose management practice . moreover , in these studies , previous treatment of diabetes was not considered , and results could have been driven by a majority of patients not previously being treated with an insulin regimen . randomized trials comparing intensive insulin therapy to more moderate approaches have reported mixed results ( 37 ) . such persisting discrepancies in the effects of intensive insulin therapy likely result from the high variability existing across centers in the multiple components of glucose control ( i.e. , monitoring , feeding , and glucose targets ) and the changes in standard practice that have occurred over time . these historic changes have modified the difference in insulin protocol between the intensive treatment and the control group in randomized clinical trials . however , in randomized controlled trials that did report a survival benefit from tight glycemic control , patients with diabetes have been the exception , demonstrating no benefit from the intervention ( 10,38 ) . our data expand this body of evidence in two crucial aspects . by considering the type of prior diabetes treatment , we show that the association between glycemic control and outcomes is different for those patients with a history of insulin therapy compared with patients without diabetes and patients with nitdm , after adjustment for baseline clinical variables . in addition , we report the economic burden associated with stress hyperglycemia for patients with and without diabetes . however , our analysis was based on an earlier , observational study ( 20 ) whose original aim was to evaluate the incidence of hospital - acquired infections after cardiac surgery , and therefore a number of limitations need to be mentioned . consequently , patients with undiagnosed diabetes may have been misclassified as not having diabetes , and for these patients diabetes control before hospital admission could not be assessed . studies ( 39 ) have reported a 10% rate of latent diabetes in icu patients with hyperglycemia . however , such misclassification , if present , would not affect the findings related to the group of patients with diabetes . second , insulin usage and parenteral nutrition protocols differed among centers , and data on adherence to these protocols were not collected . therefore , we could not include this information in our analysis . on the other hand , we adjusted for the standard insulin protocol used in the icus of the study centers , which reflects to some extent the differences in glucose management among different icus . third , the glucose measurements data , which were only collected during the 48 h after surgery ( at 6-h intervals ) , likely did not capture all the measurements that were performed in the icu after the surgery . however , such a limited sampling is unlikely to have biased the results differentially for patients with no diabetes , nitdm , and itdm . fourth , we used icd-9 codes to define complications other than infection , which did not allow us to evaluate the sequence of events that occurred , limiting conclusions about causal pathways . nevertheless , the consistency in the associations of glucose across economic and clinical outcomes further supports our conclusions . although we adjusted for a broad range of demographic characteristics and illness - related confounders , residual confounding may still have biased our findings to some extent . as is the case for observational studies , our observations only support an association between postoperative glucose levels and clinical outcomes , and not a causal relationship . likewise , we can not infer from our results whether previous insulin therapy has a causative role or is merely a marker of severity of illness associated with differential outcomes . the pandemic of diabetes calls for improved management of hyperglycemia , both outside and inside the hospital . roughly 20% of cardiac surgery patients have pre - existing diabetes , with a large proportion having more advanced disease requiring insulin therapy . more than 60% of these patients have at least one blood glucose measurement > 180 mg / dl , the glycemic threshold recommended by current clinical guidelines ( 6,40 ) . in the context of findings by others , our results support conducting a randomized controlled trial to evaluate a stratified approach to glucose control based on diabetes history and prior use of insulin . in summary , our findings suggest that current recommendations , which use a single maximum glucose threshold for the control of stress hyperglycemia after cardiac surgery , may not achieve the intended benefits in all patient subgroups . such a blanket approach could instead be harmful to patients with more advanced diabetes . given the substantial clinical and economic benefits that may be attained , patient stratification with indicators of chronic glucose dysregulation should be investigated .

this paper will explore two contrasting paradigms in mental health care and their relationship to evidence - based practice . the biomedical perspective of pathogenesis and the health perspective of salotogenesis are two major diverse views in mental health care . positive dimensions of health are traditionally viewed as software not suitable for statistical analysis , while absence of symptoms of disease are regarded as measurable and suitable for statistical analysis and appropriate as a foundation of evidence - based practice . if the main goal of mental health care is to enhance subjectively experienced health among patients , it will not be sufficient to evaluate absence of symptoms of disease as a measure of quality of care . the discussion focuses on the paradox of evidence - based absence of illness and disease versus subjectively experienced health and well - being as criterions of quality of care in mental health care . objectivethe management of postoperative hyperglycemia is controversial and generally does not take into account pre - existing diabetes . we analyzed clinical and economic outcomes associated with postoperative hyperglycemia in cardiac surgery patients , stratifying by diabetes status.research design and methodsmulticenter cohort study in 4,316 cardiac surgery patients operated on in 2010 . glucose was measured at 6-h intervals for 48 h postoperatively . outcomes included cost , hospital length of stay ( los ) , cardiac and respiratory complications , major infections , and death . associations between maximum glucose levels and outcomes were assessed with multivariable regression and recycled prediction analyses.resultsin patients without diabetes , increasing glucose levels were associated with a gradual worsening of outcomes . in these patients , hyperglycemia ( 180 mg / dl ) was associated with an additional cost of $ 3,192 ( 95% ci 1,972 to 4,456 ) , an additional hospital los of 0.8 days ( 0.4 to 1.3 ) , an increase in infections of 1.6% ( 0.5 to 2.8 ) , and an increase in respiratory complications of 2.6% ( 0.0 to 5.3 ) . however , among patients with insulin - treated diabetes , optimal outcomes were associated with glucose levels considered to be hyperglycemic ( 180 to 240 mg / dl ) . this level of hyperglycemia was associated with cost reductions of $ 6,225 ( 12,886 to 222 ) , hospital los reductions of 1.6 days ( 3.7 to 0.4 ) , infection reductions of 4.1% ( 9.1 to 0.0 ) , and reductions in respiratory complication of 12.5% ( 22.4 to 3.0 ) . in patients with non insulin - treated diabetes , outcomes did not differ significantly when hyperglycemia was present.conclusionsglucose levels < 180 mg / dl are associated with better outcomes in most patients , but worse outcomes in patients with diabetes with a history of prior insulin use . these findings support further investigation of a stratified approach to the management of patients with stress - induced postoperative hyperglycemia based on prior diabetes status .

gastroesophageal reflux disease ( gerd ) is a common gastrointestinal disease in asia as well as in the west ( 1 , 2 ) . studies have reported that 10%-20% of the adult western population experience typical gerd symptoms ( heartburn and/or regurgitation ) at least once per week ( 1 ) . the prevalence of gerd in adult koreans is reported to be 3.5%-8.5% ( 3 - 5 ) . this increase has been possibly attributed to changes in diet , an increasing aged population , the increasing frequency of endoscopic examinations , and the widely spreading knowledge on gerd . the montreal definition describes gerd as a condition that develops when the reflux of gastric contents causes troublesome symptoms and/or complications ( 6 ) . a number of regional and international guidelines and recommendations for the management of gerd have been published . the genval workshop report on reflux disease management , published in 1999 ( 7 ) , and the asia - pacific consensus on the management of gerd , published in 2008 ( 8) , previous studies show that proton pump inhibitors ( ppis ) are the most effective drug for patients with gerd ( 7 , 8) . actually , ppis are known to be most commonly prescribed for the treatment of gerd . however , the prescription pattern of ppis in patients with gerd may be different between countries , which is partly attributed to the economic situation or the health insurance system . in korea , national health insurance covers the cost for the medication prescribed in hospitals and clinics according to the insurance reimbursement guideline . accordingly , the prescription pattern of ppis used for the treatment of gerd is substantially influenced by the insurance reimbursement guideline . thus , the aim of this study was to document practice pattern of gastroenterologists for the management of gerd patients under the minimal influence of the insurance reimbursement guideline . fifty - six gastroenterologists at 51 secondary and tertiary care hospitals representing most regions of korea participated in the study . all participating gastroenterologists were required to recruit about 20 consecutive eligible patients who complained of typical gerd symptoms ( heartburn and/or regurgitation ) . patients were eligible for inclusion in this study if they were aged 18 yr and had typical gerd symptoms ( heartburn and/or regurgitation ) at least once per week over the previous 7 days with or without esophagitis . patients were excluded from participation if they had taking ppis or histamine 2 receptor blocking agents within the previous 4 weeks . the other exclusions were hypersensitivity to the active ingredient of ppis and the presence of organic diseases such as gastrointestinal cancer , severe liver disease , pancreatic disease , peptic ulcer , inflammatory bowel disease , severe renal impairment , or severe respiratory disease . thus , we tried to reduce the influence of the study on the routine clinical practice for enrolled patients . the only thing that was affected by the study protocol was to choose a ppi . in order to minimize the influence of the insurance reimbursement guideline on the use of ppis , we recommended the use of rabeprazole ( pariet , janssen korea ltd . , seoul , korea ) for patients in whom ppi treatment should be considered for the management of gerd symptoms , because rabeprazole is the only ppi of which both full - dose and half - dose are permitted for the initial and maintenance treatment of gerd , irrespective of the presence of erosive esophagitis , in the national insurance reimbursement guideline . the others associated with the treatment for patients enrolled in the study had not been set by the study protocol . , gastroenterologists followed their usual practice of patient care ; the severity of symptoms and medications prescribed were recorded at each visit , using a web - based electronic data capture system . at the visit for inclusion , demographic data and information on the patient 's symptoms , previous investigations for gerd , and treatment received were documented . in addition , the frequency ( days / week ) and severity ( 0 , none ; 1 , mild ; 2 , moderate ; 3 , severe ) of symptoms over the previous 7 days were assessed . when esophagogastroduodenoscopy ( egd ) or 24-hr esophageal ph monitoring had been performed for the evaluation of gerd symptoms , its results were reviewed . since this study was not a clinical trial , there was no hypothesis for the determination of the adequate sample size . nonetheless , a sample size of 1,120 patients ( 20 patients per a gastroenterologist ) was planned . a subgroup analysis was carried out using the chi - squared test and student 's t - test . the statistical analysis was performed using spss for windows version 11 ( spss inc . , the study was approved by each of the institutional review boards of the hospitals where the principal investigator ( approval number : 4 - 2008 - 0201 ) and other investigators belonged . each patient received information on the study design such as data handling and aims of the study . this study was an observational study and designed to have as little impact as possible on the management of patients . the study was approved by each of the institutional review boards of the hospitals where the principal investigator ( approval number : 4 - 2008 - 0201 ) and other investigators belonged . each patient received information on the study design such as data handling and aims of the study . this study was an observational study and designed to have as little impact as possible on the management of patients . fifty - six gastroenterologists at 32 secondary and 19 tertiary care hospitals participated in the study . a total of 1,197 patients ( 523 males , 44% ) of mean age 53.1 ( 13.6 ) yr were recruited . fifty - seven percent of them were enrolled at secondary care hospitals and the others ( 43% ) at tertiary care hospitals . at the time of enrollment , 479 patients ( 40% ) had previous egd results and 382 patients ( 32% ) underwent egd before the start of medications . thirty - six percent of those who had egd results showed erosive esophagitis , and 3% had barrett 's esophagus . at the time of enrollment , 6 patients had previous results of 24-hr esophageal ph monitoring . seven patients underwent 24-hr esophageal ph monitoring during the evaluation period before the start of medications , all of whom had atypical gerd symptoms and history of previous treatment for gerd symptoms . baseline data and symptoms on patients enrolled in the study are shown in table 1 and 2 , respectively . the most common accompanying symptom was chest pain , followed by postprandial fullness , globus , belching , nausea , abdominal pain , early satiety , sore throat , chronic cough , hoarseness , dysphagia , vomiting , and odynophagia in the order of prevalence . during the 16-week study period , the mean total number of visits per patient was 3.4 ( 1.4 , ranged 1 - 6 ) . the total duration of ppi treatment during the study period was 2 weeks in 2% , > 2 weeks and 4 weeks in 9% , > 4 weeks and 8 weeks in 10% , > 8 weeks and 12 weeks in 5% , and > 12 weeks and 16 weeks in 74% . the total number of visits and the total duration of ppi treatment did not significantly differ between patients with and without erosive esophagitis ( table 3 ) . at the start of treatment , gastroenterologists prescribed rabeprazole 20 mg ( full - dose ) daily to 94% of the patients who received ppi treatment and rabeprazole 10 mg ( half - dose ) daily to the remaining 6% . at the second visits , rabeprazole 20 mg daily was prescribed to 89% of those who were followed , and 10 mg daily to the remainder . at the third visit , the participating gastroenterologists prescribed rabeprazole 20 mg daily to 70% of those who were followed and 10 mg daily for the remaining 30% . during ppi treatment , prokinetics , mucosal protectives , antacids and histamine 2 receptor blocking agents were concomitantly used in 31% , 5% , 4% , and 1% of the patients , respectively . fifty - six gastroenterologists at 32 secondary and 19 tertiary care hospitals participated in the study . a total of 1,197 patients ( 523 males , 44% ) of mean age 53.1 ( 13.6 ) yr were recruited . fifty - seven percent of them were enrolled at secondary care hospitals and the others ( 43% ) at tertiary care hospitals . at the time of enrollment , 479 patients ( 40% ) had previous egd results and 382 patients ( 32% ) underwent egd before the start of medications . thirty - six percent of those who had egd results showed erosive esophagitis , and 3% had barrett 's esophagus . at the time of enrollment , 6 patients had previous results of 24-hr esophageal ph monitoring . seven patients underwent 24-hr esophageal ph monitoring during the evaluation period before the start of medications , all of whom had atypical gerd symptoms and history of previous treatment for gerd symptoms . baseline data and symptoms on patients enrolled in the study are shown in table 1 and 2 , respectively . the most common accompanying symptom was chest pain , followed by postprandial fullness , globus , belching , nausea , abdominal pain , early satiety , sore throat , chronic cough , hoarseness , dysphagia , vomiting , and odynophagia in the order of prevalence . during the 16-week study period , the mean total number of visits per patient was 3.4 ( 1.4 , ranged 1 - 6 ) . the total duration of ppi treatment during the study period was 2 weeks in 2% , > 2 weeks and 4 weeks in 9% , > 4 weeks and 8 weeks in 10% , > 8 weeks and 12 weeks in 5% , and > 12 weeks and 16 weeks in 74% . the total number of visits and the total duration of ppi treatment did not significantly differ between patients with and without erosive esophagitis ( table 3 ) . at the start of treatment , gastroenterologists prescribed rabeprazole 20 mg ( full - dose ) daily to 94% of the patients who received ppi treatment and rabeprazole 10 mg ( half - dose ) daily to the remaining 6% . at the second visits , rabeprazole 20 mg daily was prescribed to 89% of those who were followed , and 10 mg daily to the remainder . at the third visit , the participating gastroenterologists prescribed rabeprazole 20 mg daily to 70% of those who were followed and 10 mg daily for the remaining 30% . during ppi treatment , prokinetics , mucosal protectives , antacids and histamine 2 receptor blocking agents were concomitantly used in 31% , 5% , 4% , and 1% of the patients , respectively . several international guidelines for the management of gerd are reported ( 6 - 8 ) , but no domestic guidelines on clinical practice for patients with gerd have been issued yet in korea . in the present study , we followed patients with typical gerd symptoms for 16 weeks , and observed gastroenterologists ' treatment patterns . the current study showed that ppis are prescribed for the majority of patients with typical gerd symptoms and that korean gastroenterologists preferred a full - dose ppi for both initial and maintenance treatment of gerd , irrespective of the presence of erosive esophagitis , rather than a half - dose ppi . primary care clinics were excluded , because the aim of the study was to document gastroenterologists ' treatment patterns . patients with typical gerd symptoms who visit secondary and tertiary care hospitals participating in the study are treated by gastroenterologists . the institutions involved were selected on a nationwide scale in consideration of the number of regional population . egd is relatively cheap in korea because its cost is covered by national medical insurance . seventy - two percent of patients enrolled in the study had the results of egd . erosive esophagitis was observed in 36% of them , the majority of which was of grade la - a or la - b . longitudinal studies conducted in asia show an increase in the prevalence of gerd symptoms and reflux esophagitis ( 9 - 11 ) . the ratio of erosive reflux disease ( erd ) to nonerosive reflux disease ( nerd ) in asia is believed to be lower than in the west ( 12 ) . the ratio of erd found in the present study was 36% , which seems to be getting comparable to that reported in the west . this may be partly attributed to the fact that the participating institutions were secondary and tertiary care hospitals . since studies on the sensitivity and specificity of heartburn for predicting the presence of gerd are lacking in korea , it is uncertain whether heartburn is a definite marker of gerd in the korean population . functional heartburn is believed to be a different entity from nerd , particularly in terms of acid reflux patterns and the responsiveness to ppis ( 13 ) . functional heartburn can be diagnosed when there are no abnormal acid or nonacid esophageal reflux , no symptom correlation with reflux , and no response to ppis ( 14 ) . abnormal acid or nonacid reflux can be determined using a 24-hr esophageal ph / impedance monitoring . esophageal ph monitoring was available in 28 institutions ( 55% of the participating institutions ) . however , only 1% of the study patients underwent 24-hr ambulatory esophageal ph monitoring , which suggests that this test is not commonly performed even at secondary and tertiary care hospitals . in general , ppi trial is recommended to be preceding esophageal ph monitoring , particularly in patients with typical gerd symptoms . in the present study , ambulatory esophageal ph monitoring was performed mainly in patients with atypical gerd symptoms and history of previous ppi treatment for gerd symptoms , which is in keeping with the current guidelines . the asian - pacific experts for gerd reported a general consensus that ppis provide the most effective treatment for erd and nerd ( 8) . the consensus is based on previous studies showing that ppis are superior to histamine 2 receptor blocking agents in terms of healing esophageal mucosa and relieving heartburn symptoms in patients with gerd ( 15 - 17 ) . in the present study , most of patients enrolled in the study received ppi treatment ( 87% of the study patients ) . prokinetics were the most commonly used concomitant medications , which can be possibly supported by the positive value of prokinetics in the treatment of gerd ( 18 , 19 ) and substantial overlap between gerd symptoms and dyspepsia ( 5 , 20 ) . our findings of the present study that 27% and 15% of the patients with typical gerd symptoms had postprandial fullness and early satiety , respectively , demonstrate overlap between gerd symptoms and dyspepsia . the asian - pacific consensus includes statements on the duration of ppi treatment for erd and nerd as follows ; " nerd patients require more than 4 weeks of initial continuous ppi therapy and erd patients need a minimum of 4 - 8 weeks of initial continuous ppi therapy " ( 8) . it is difficult to determine timing to change initial treatment to maintenance treatment . according to the guidelines , maintenance treatment usually begins after 4 weeks of initial treatment in nerd patients and after 8 weeks of initial treatment in patients with erd . the influence of national health insurance system on the use of drugs is too big in korea , because it covers all patients visiting hospitals due to gerd symptoms . based on the national health insurance reimbursement guideline , full - dose of most ppis , except rabeprazole , is permitted only for the initial treatment of erd , but not for the initial treatment of nerd and the maintenance treatment of erd and nerd . according to that guideline , only half - dose ppi can be used for the initial treatment in patients with gerd symptoms who have no endoscopic findings and in nerd patients , and for the maintenance treatment of gerd . rabeprazole is the only ppi of which both full - dose and half - dose are permitted for the initial and maintenance treatment of erd and nerd under the korean health insurance system . so , in order to reduce distortion or variability in the clinical practice for gerd patients due to the national health insurance reimbursement guideline , we made a decision to recommend rabeprazole when ppi treatment is required for the enrolled patients . since our main aims were to investigate the prescription pattern on the dosage of ppis used for the treatment of gerd and to know whether korean gastroenterologists keep the international guideline for the treatment of gerd , we tried to reduce the influence of the national health insurance reimbursement guideline and the study protocol . thus , treatment for patients enrolled in the study had not been set by the study protocol , and participating gastroenterologists carried out their practice at their own decision . given that rabeprazole 20 mg daily was used as initial treatment in 94% of the patients who received ppi treatment , the majority of korean gastroenterologists appear to prefer a full - dose ppi for the initial treatment of gerd , irrespective of the presence of erosive esophagitis . gastroenterologists participating in this study tended to use a full - dose ppi without step - down of the dose . these points are not in keeping with the insurance reimbursement criteria , which might be attributed to a high proportion of gerd patients requiring a long - term treatment of a fulldose ppi . in the present study , the mean duration of continuous ppi therapy during the 16-week observational period was 96 and 95 days in patients with and without erosive esophagitis , respectively . in the present study , 62% of the study patients were continuously treated with ppis until the end of the study period . participating gastroenterologists were allowed to prescribe only rabeprazole , either 10 mg or 20 mg , if they want to use ppi . rabeprazole is the only ppi that both dosages are permitted to be used in the maintenance therapy according to the reimbursement guideline . accordingly , they appear to choose the ppi dosage mainly based on the treatment efficacy . if other ppis would have selected for this study , they might have prescribed according to the reimbursement guideline , irrespective of the treatment efficacy . one of our aims was to investigate drug compliance , but we found that drug compliance was impossible to investigate , because many patients were not followed during the study period . since this was an observational study , follow - up was not controlled by the study protocol . so , follow - up loss was not prevented , and drug compliance could not be investigated . according to the study design , participating gastroenterologists were able to make a decision for tests and treatment for the enrolled patients at their discretion . thus , our findings suggest that many korean patients with typical gerd symptoms need a long - term treatment of a full - dose ppi . in conclusion , endoscopy is commonly performed for the evaluation of gerd symptoms , but 24-hr esophageal ph monitoring is not . a full - dose ppi is preferred for the initial and maintenance treatment of gerd under the minimal influence of the insurance reimbursement guideline , which may reflect a high proportion of gerd patients requiring a long - term treatment of a full - dose ppi . practice pattern of gastroenterologists for the management of gerd under the minimal influence of the insurance reimbursement guideline : a multicenter prospective observational study kwang jae lee , jin il kim , ju sang park , byung sik moon , sang - gyun kim , jae hee chun , hoon - yong jung , chang hwan choi , seong woo chun , geun am song , myung gyu choi and hoon jai chun we studied practice pattern of gastroenterologists for the management of gerd under the minimal influence of the insurance reimbursement guideline in korea . a full - dose proton pump inhibitor ( ppi ) is preferred for the initial and maintenance treatment of gerd , which may reflect a high proportion of gerd patients requiring a long - term treatment of a full - dose ppi . a case report of a 56-year - old male farmer who sought medical attention after a month - long evolution of irritative symptoms in his right eye , accompanied by visual acuity ( va ) impairment . the patient received topical and oral broad - spectrum antibiotic treatment with no improvement before being referred to a cornea specialist , where he was found to have va of 20/150 and was noted on biomicroscopy to have endothelial feathery coalescent lesions . the patient was admitted to the hospital for an aqueous humor sample and intravenous voriconazole . however , clinical evidence of improvement was confirmed after 5 days of antimycotic intravenous therapy . complete clinical resolution was achieved at 1 month after treatment completion with oral voriconazole , as evidenced by va of 20/20 and disappearance of endothelial lesions . endothelial involvement by fungi is a rare condition . in this case , no microbes were isolated , but the characteristic morphology of the lesions , the history of onychomycosis , and the spectacular response to voriconazole turn this case into a valid presumptive diagnosis . the cornea is the ocular structure most commonly affected by fungi . most often , the source of infection is exogenous , such as vegetal trauma , among others . in addition , it is associated with any disease affecting the eye s normal surface , such as persistent epithelial defects , neurotrophic ulcers , use of contact lenses , and secondary attenuation of cell defense mechanisms after topical steroids.15 infectious endothelial involvement of the cornea has been mainly attributed to different viral species.6 however , herein we present the case of a patient with a clear endothelial infectious involvement that successfully responded to systemic antifungal therapy with voriconazole . a 56-year - old male banana grower sought medical attention after a month - long evolution of irritative symptoms in his right eye accompanied by visual acuity ( va ) impairment . his medical history confirmed excision of bilateral pterygium 15 years earlier , but no recent ocular trauma was related by the patient . the general medical practitioner ( nonophthalologist ) initiated treatment with topical prednisolone acetate 1% and gentamicin sulfate 0.3% every 6 hours for 1 week for a presumed bacterial conjunctivitis , but the patient was referred to the ophthalmologist after lack of success with this therapy . an initial diagnosis of infectious keratitis ( with unaffected epithelium but endothelial involvement ) was made , and treatment with topical moxifloxacin chlorhydrate 0.5% four times per day and gentamicin sulfate 0.3% three times per day plus oral ciprofoxacin 750 mg every 12 hours was prescribed . after 6 days on this therapeutic regimen and with lack of clinical improvement , the patient was referred to a cornea specialist . he was found with right eye va of 20/150 and normal va in his left eye . the biomicroscopy performed on the affected eye confirmed the integrity of the epithelium with an unruptured bulla over the pupillary area , presence of folds on descemet s membrane , anterior chamber cells 2+(standardization of uveitis nomenclature working group),7 and multiple coalescent circular lesions of feathery and whitish appearance on the endothelium ( figure 1a ) . fundoscopic examination with an indirect ophthalmoscope under midriasis was normal . based on these findings , therefore , the patient was admitted to hospital to further explore the etiology of the lesion . for this , an aqueous humor sample was examined for fungi detection by culture and polymerase chain reaction ( pcr ) , and systemic antimycotic therapy was initiated immediately after sample taking . by recommendation of the infectologist , the patient was empirically treated with voriconazole 200 mg intravenously ( iv ) every 12 hours ( diluted in 250 ml of 0.9% saline solution administrated over 2 hours ) and topical gatifloxacin 0.3% every 6 hours for 1 week ( as post puncture antibacterial prophylaxis ) . after 3 days on iv therapy with voriconazole , the patient showed significant clinical improvement , evidenced by an important decline in the number of cells within the anterior chamber and some reduction on the feathery endothelial lesions described previously . aqueous humor microbiologic study was negative ( gram , potassium hydroxide staining , aerobic , anaerobic , and fungal cultures ) . pcr , venereal disease research laboratory , herpes simplex , and hiv testing were also negative . after 5 days on this regimen , the patient was discharged with voriconazole 200 mg orally every 12 hours . after a week , the patient was found to have va of 20/30 , considerable improvement of endothelial lesions , and faint residual corneal edema . the same treatment regimen was administered for a further week , and fluorometholone 0.1% every 12 hours was added to therapy for edema . examination 2 weeks after discharge confirmed further edema reduction and almost complete resolution of endothelial lesions . after 4 weeks of antifungal treatment ( 3 weeks of those were as an outpatient ) , the patient showed full edema resolution , the presence of a few small endothelial scars , and 20/20 va ( figure 1c ) . fluorometholone was then withheld and oral voriconazole was continued until completion of 2 months with antifungal therapy . infectious endothelial involvement of the cornea has been mainly attributed to different viral species.6 however , we report an interesting case of a patient whose endothelial lesions suggest mycotic etiology that successfully responded to systemic antifungal therapy with voriconazole . usual manifestations of fungal keratitis include epithelial and stromal ulceration , abscessing with stromal necrosis , epithelial or stromal edema and , rarely , cell reaction in the anterior chamber , hypopyon , and fibrin deposits within the anterior chamber and endothelial plates.1,5 nevertheless , the most striking finding in this case was the confinement of damage to endothelial plates with subsequent central corneal edema , bulla formation , folds on descemet s membrane , and cell reaction in the anterior chamber . therefore , we propose an endogenous source of infection , and postulate the presence of recurrent onychomycosis as a risk factor in the absence of prior trauma or epithelial disruption . however , microbiological isolates turned out to be negative both in blood and in aqueous humor . a characteristic clinical sign of fungal keratitis is the irregular nature of infiltrates , traditionally described as feathery or cotton wool.1,4,8 this feature prompted the suspicion of fungal origin in our patient , as this pattern was observed on the endothelium . although gram and potassium hydroxide staining , as well as pcr , were negative , our diagnosis was established on clinical grounds and confirmed by the success of empirical therapy with voriconazole . it is important to point out that laboratory diagnosis in this case has been limited by the low volume of sample taken , which was destined for multiple studies ( stains , cultures , pcr ) . in addition , experience with these tests and available primers in our country are also limited . some authors have reported on the good efficacy of intracameral injection with amphotericin b and voriconazole in cases of endophthalmitis secondary to keratitis.911 although this is an adequate therapeutic alternative , it was not required , as the patient showed an excellent response to systemic therapy with oral voriconazole . an interesting case of a presumed endogenous mycotic endotheliitis is herein reported in a patient with a history of recurrent onychomycosis who develops endothelial lesions of mycotic morphology . the patient responds to a systemic iv and oral scheme with voriconazole , and thus an endogenous and mycotic etiology is presumed . however , this could not be confirmed by microbiological studies , due to the limitations that we have cited .

the objective of the study was to document practice pattern of gastroenterologists for the management of gastroesophageal reflux disease ( gerd ) under the minimal influence of the insurance reimbursement guideline . data on management for 1,197 consecutive patients with typical gerd symptoms were prospectively collected during 16 weeks . in order to minimize the influence of reimbursement guideline on the use of proton pump inhibitors ( ppis ) , rabeprazole was used for the ppi treatment . a total of 861 patients ( 72% ) underwent endoscopy before the start of treatment . ppis were most commonly prescribed ( 87% ) . at the start of treatment , rabeprazole 20 mg daily was prescribed to 94% of the patients who received ppi treatment and 10 mg daily to the remaining 6% . at the third visits , rabeprazole 20 mg daily was prescribed to 70% of those who were followed and 10 mg daily for the remaining 30% . continuous ppi treatment during the 16-week period was performed in 63% of the study patients . in conclusion , a full - dose ppi is preferred for the initial and maintenance treatment of gerd under the minimal influence of the insurance reimbursement guideline , which may reflect a high proportion of gerd patients requiring a long - term treatment of a full - dose ppi . purposeto report an interesting case of infectious endotheliitis of presumed mycotic origin.methodsa case report of a 56-year - old male farmer who sought medical attention after a month - long evolution of irritative symptoms in his right eye , accompanied by visual acuity ( va ) impairment . the patient received topical and oral broad - spectrum antibiotic treatment with no improvement before being referred to a cornea specialist , where he was found to have va of 20/150 and was noted on biomicroscopy to have endothelial feathery coalescent lesions . the patient was admitted to the hospital for an aqueous humor sample and intravenous voriconazole.resultsthe microbiological studies did not isolate any micro - organisms . however , clinical evidence of improvement was confirmed after 5 days of antimycotic intravenous therapy . complete clinical resolution was achieved at 1 month after treatment completion with oral voriconazole , as evidenced by va of 20/20 and disappearance of endothelial lesions.conclusionendothelial involvement by fungi is a rare condition . in this case , no microbes were isolated , but the characteristic morphology of the lesions , the history of onychomycosis , and the spectacular response to voriconazole turn this case into a valid presumptive diagnosis .

the experimental animals , feeding and management : the experimental procedures complied with the guide for the care and use of agricultural animals of obihiro university . the experiment was carried out at the field center of animal science and agriculture , obihiro university of agriculture and veterinary medicine . fifty multiparous holstein cows , in which parity was from 1st to 5th at dry period , were used in this study and had calved between september 2011 and august 2012 . parity and body condition score ( bcs ) of the experimental cows at initiation of the study were 2.4 0.2 and 3.41 0.04 , respectively . the study was performed from 3 weeks before the expected parturition to 100 days pp . cows close to the dry period , about 1 month before the expected calving date , were moved to a paddock and fed a limited total mixed ration [ dry matter ( dm ) basis : 127 g of crude protein ( cp)/kg and 6.6 mj of net energy for lactation ( nel)/kg ] consisting of grass silage ( 3.5 kg , dm basis : 165 g of cp / kg and 5.5 mj of nel / kg ) , maize silage ( 5.1 kg , dm basis : 86 g of cp / kg and 6.0 mj of nel / kg ) , concentrate for dry cows ( 2.0 kg , dm basis : 170 g of cp / kg and 6.8 mj of nel / kg ) and grass hay ( dm basis : 125 g of cp / kg and 5.7 mj of nel / kg ) ad libitum until parturition . after parturition , cows were housed in a free - stall barn and fed a lactation diet , which was a mixed ration ( dm basis : 155 g of cp / kg and 6.2 mj of nel / kg ) consisting of grass ( 6.5 kg , dm basis : 165 g of cp / kg and 5.5 mj of nel / kg ) , maize silage ( 12.5 kg , dm basis : 84 g of cp / kg and 6.4 mj of nel / kg ) and concentrate for dairy cows ( 8.0 kg , dm basis : 180 g of cp / kg and 7.1 mj of nel / kg ) ad libitum . in addition , the diets were supplemented with minerals , and the dairy cow concentrate was prepared according to each cow s specific requirements for milk production . grass hay ( dm basis : 104 g of cp / kg and 5.2 mj of nel / kg ) and water were available ad libitum . cows were milked twice daily between 05:00 and 06:30 hr and between 17:00 and 18:30 hr . the experimental itt and sampling : the itt was performed 3 weeks before the expected calving date . the cows were weighed the day before the initiation of the itt , and bw was used to determine the doses of insulin for the itt . immediately before the itt , an extension catheter was inserted into the right or left jugular vein . the itt was performed by intravenously administering 0.05 iu / kg bw of insulin ( novolin r 100 iu / ml ; novo nordisk pharma , tokyo , japan ) , followed by administration of 5 ml heparinized saline ( 100 iu / ml ) . blood samples were collected via the jugular vein at 0 ( before insulin injection ) , 30 , 45 and 60 min relative to the administration of insulin via caudal venipuncture to measure glucose and insulin . bcs was assessed twice a week from 3 weeks before the expected parturition to 3 weeks after calving by the same operator by using a 1 to 5 scale with 0.25 intervals , where 1=thin and 5=very fat . blood samples were obtained by caudal venipuncture twice a week from 3 weeks before the expected parturition to 3 weeks after calving . blood samples were collected via the jugular vein from the calves immediately after birth . nonheparinized and silicone - coated 9-ml tubes ( venoject , autosep , gel + clot . act . vp - as109k ; terumo corporation , tokyo , japan ) were used for biochemical analysis , and sterile 10-ml tubes containing 200 l of stabilizer solution ( 0.3 m edta and 1% acetyl salicylic acid , ph 7.4 ) were used for hormonal analysis . serum was obtained by centrifuging the blood samples for 15 min at 38c in an incubator . all the tubes were centrifuged at 2,000 g for 20 min at 4c , and plasma samples were maintained at 30c until analysis . in addition , milk samples were collected twice a week after milking until the onset of luteal activity . the milk samples were centrifuged at 1,500 g for 15 min at 4c , and the skim milk samples were stored at 30c until analysis for progesterone concentration . daily milk yield was recorded until 100 days pp . peripartum diseases , such as milk fever , hypocalcemia , ketosis , ruminal acidosis , displaced abomasum , lameness , retained placenta , endometritis and mastitis , were recorded when that has been diagnosed from 3 weeks prepartum to 3 weeks postpartum by veterinarian in the experimental farm . the experimental measurement of hormones and metabolites : plasma and skim milk progesterone concentrations were determined using enzyme immunoassay ( eia ) after extraction with diethyl ether , as described previously ; the extraction efficiency was 90% . the standard curve ranged from 0.05 to 50 ng / ml , and the 50% effective dose ( ed50 ) of the assay was 0.66 ng / ml . the mean intra - assay and inter - assay coefficients of variation ( cvs ) were 6.0% and 9.2% , respectively . the total plasma insulin - like growth factor 1 ( igf-1 ) concentration was determined using eia by using the biotin streptavidin amplification technique after protein extraction by using acid ethanol ( 87.5% ethanol and 12.5% 2 n hydrochloric acid ) to obtain igf-1 free from binding proteins . intra- and inter - assay cvs were 5.9% and 6.1% , respectively , and the ed50 of this assay system was 7.2 ng / ml . the plasma gh concentrations were determined using eia as described previously ; the standard curve ranged from 0.78 to 100 ng / ml , and the ed50 was 21 ng / ml . intra- and inter - assay cvs were 3.1% and 8.2% , respectively . the plasma insulin concentrations were determined using an enzyme - linked immunosorbent assay ( elisa ) kit ( bovine insulin elisa 10 - 1201 - 01 ; mercodia , uppsala , sweden ) . the serum concentrations of glucose , non - esterified fatty acids ( nefa ) , -hydroxybutyrate ( bhba ) , total protein ( tp ) , albumin ( alb ) , blood urea nitrogen ( bun ) and total cholesterol ( t - cho ) and the activities of aspartate aminotransferase ( ast ) were measured using a clinical chemistry automated analyzer ( tba120fr ; toshiba medical systems co. , ltd . , tochigi , japan ) . the experimental identification of the onset of luteal activity : when the progesterone concentration in the plasma or skim milk had increased to more than 1 ng / ml , the cows were considered to show luteal activity . the experimental statistical analysis : sixteen cows were excluded from data analysis , because of the following reasons : a pregnancy period of more than 287 or less than 273 days ( n=6 ) , severe mastitis ( n=3 ) , twin calving ( n=1 ) , blood collection loss at itt ( n=3 ) and mistakes in insulin injection at itt ( n=3 ) . cows were divided into two groups based on the time required for glucose to reach the minimum levels after insulin injection . cows with a minimum glucose at 60 min after insulin injection were considered to have lower insulin sensitivity and/or lower glucose metabolism compared to cows with a minimum glucose level by 45 min after insulin injection . therefore , cows with a minimum glucose level at 60 min after insulin injection were defined as the insulin resistant group ( ir group ) , whereas those with a minimum glucose level by 45 min after insulin injection were defined as the non - insulin resistant ( nir group ) in this study . before data analysis , bcs , plasma igf-1 , gh and insulin concentrations , and serum metabolite concentrations were averaged weekly . the period of 06 days after calving was considered as the parturient week ( 0 week pp ) , and the kolmogorov smirnov test ( sas enterprise guide version 4.3 ; sas institute inc . , cary , nc , u.s.a . ) was used for statistical testing of normality . in addition , the data were analyzed separately for the prepartum and pp periods . stat view ( stat view 5.0 software ; abacus concepts inc . , berkeley , ca , u.s.a . ) was used for data analysis by using the repeated measures of the anova procedure , including time ( week ) , group ( nir or ir ) and their interaction in the model as fixed effects . diagnosis of peripartum diseases and sex of calves in the nir and ir groups were analyzed using the chi - square test , and other data , including results for calves between nir and ir , were analyzed using the student s t - test or wilcoxon s signed rank test ( sas enterprise guide version 4.3 ; sas institute inc . ) . results are presented as mean standard error of the mean ( sem ) ; differences with p<0.05 were considered significant . in 28 of the 34 experimental cows , the time required for glucose to reach the minimum level was 45 min after insulin injection with one exception ( 30 min ; n=1 , 45 min ; n=27 , nir group ) . the remaining experimental cows ( n=6 ) required 60 min after insulin injection to attain the minimum glucose levels ( ir group ) . serum glucose concentrations at 60 min after insulin injection were higher in the nir group than in the ir group , although glucose levels at the other time points did not differ between the nir and ir groups ( fig . 1.the change in serum glucose concentration after insulin injection at insulin tolerance test ( itt ) in the nir ( n=28 ) and ir ( n=6 ) groups . p<0.05 ) . the change in serum glucose concentration after insulin injection at insulin tolerance test ( itt ) in the nir ( n=28 ) and ir ( n=6 ) groups . table 1table 1.parity , calving difficulty , sex of calves , peripartum disease , luteal activity onset and milk yield in the nir and ir groupsnir groupir groupp - value(n=28)(n=6)parity at the onset of experiment2.4 0.32.2 0.70.460calving difficulty1.1 0.11.0 0.00.700sex of calves ( male / female)14/143/31.000diagnosis of peripartum disease9/28 ( 32%)1/6 ( 17%)0.645days to the onset of luteal activity ( days)38.3 3.820.3 3.60.039average of daily milk yield between days 7 and 100 pp ( kg)41.4 0.935.9 2.00.013total milk yield from days 7 to 100 pp ( kg)3,888.1 81.23,375.5 185.90.013values are the mean sem . a ) nir group ; cows with a minimum glucose level by 45 min after insulin injection . ir group ; cows with a minimum glucose level at 60 min after insulin injection . b ) 1 , unassisted birth ( natural , without human assistance ) ; 2 , easy calving with human assistance ; 3 , difficult calving with a few humans ; 4 , dystocia ( requiring considerably more force than normal ) ; and 5 , surgical treatment or death of cow . c ) milk fever , hypocalcemia , ketosis , ruminal acidosis , displaced abomasum , lameness , retained placenta , endometritis and mastitis from 3 weeks prepartum to 3 weeks postpartum . shows the parity , calving difficulty , sex of calves , peripartum disease diagnosis , luteal activity onset and milk yield until 100 days pp in the nir and ir groups . days until the onset of luteal activity in the ir group were fewer than those in the nir group ( p<0.05 ) . in addition , the average ( p<0.05 ) and total ( p<0.05 ) milk yields until 100 days pp were lower in the ir group than in the nir group . peripartum diseases were diagnosed as mastitis ( n=6 ) , hypocalcemia ( n=1 ) and milk fever ( n=2 ) in nir group , and as mastitis ( n=1 ) in ir group , and there was no significant difference in the number of cows with the peripartum diseases between nir and ir groups . no significant difference was noted in other factors between the nir and ir groups . a ) nir group ; cows with a minimum glucose level by 45 min after insulin injection . ir group ; cows with a minimum glucose level at 60 min after insulin injection . b ) 1 , unassisted birth ( natural , without human assistance ) ; 2 , easy calving with human assistance ; 3 , difficult calving with a few humans ; 4 , dystocia ( requiring considerably more force than normal ) ; and 5 , surgical treatment or death of cow . c ) milk fever , hypocalcemia , ketosis , ruminal acidosis , displaced abomasum , lameness , retained placenta , endometritis and mastitis from 3 weeks prepartum to 3 weeks postpartum . 2.serum metabolite concentrations , activities of enzymes and plasma metabolic hormones , and bcs during the experimental period [ mean sem : solid , nir ( n=28 ) ; open , ir ( n=6 ) groups ] . * indicates differences of p<0.05 , and indicates differences of p<0.1 between the nir and ir groups . shows the circulating serum metabolite concentrations , enzyme levels , plasma metabolic concentrations and bcs during the experimental period . during the prepartum period , bcs ( p<0.05 ) , and serum bun concentrations ( p<0.05 ) were lower , whereas serum glucose ( p=0.05 ) and alb concentrations ( p=0.10 ) tended to be lower in the ir group than in the nir group . during the pp period , cows of the nir group had higher serum nefa ( p<0.05 ) and bhba ( p=0.09 ) concentrations than those in the ir group . in addition , treatment and time effects were observed ( p<0.05 ) for bcs during the pp period : bcs at 0 ( p=0.08 ) and 1 ( p<0.05 ) week pp were lower in the ir group than in the nir group . no significant differences were noted in the other factors between the nir and ir groups in each period . serum metabolite concentrations , activities of enzymes and plasma metabolic hormones , and bcs during the experimental period [ mean sem : solid , nir ( n=28 ) ; open , ir ( n=6 ) groups ] . * indicates differences of p<0.05 , and indicates differences of p<0.1 between the nir and ir groups . bw , plasma metabolic hormone levels and serum glucose concentrations at birth in the calves of cows of the nir and ir groups are shown in table 2table 2.bw and plasma metabolic hormones and serum glucose concentrations at birth in the calves of the nir and ir groupscalves of nircalves of irp - value(n=28)(n=6)bw at the birth ( kg)47.2 0.942.1 1.70.020plasma gh concentration ( ng / ml)13.6 1.315.2 4.80.653plasma igf-1 concentration ( ng / ml)121.5 6.369.8 5.60.001plasma insulin concentration ( ng / ml)0.3 0.00.7 0.20.061serum glucose concentration ( mg / dl)77.4 5.272.1 14.80.684values are the mean sem . a ) nir group ; cows with a minimum glucose level by 45 min after insulin injection . ir group ; cows with a minimum glucose level at 60 min after insulin injection .. bw at birth in the calves of the ir group was lower than that in the calves of the nir group ( p<0.05 ) . furthermore , the calves of the ir group showed lower plasma igf-1 concentration ( p<0.001 ) and higher plasma insulin concentration ( p=0.06 ) . no significant differences were noted in the plasma gh and serum glucose levels at birth between the calves of the nir and ir groups . a ) nir group ; cows with a minimum glucose level by 45 min after insulin injection . ir group ; cows with a minimum glucose level at 60 min after insulin injection . in this study , the six cows that reached the minimum glucose levels at 60 min after insulin injection were considered to be ir ; the reason for ir was thought to be the slow recovery of glucose after insulin injection , which is consistent with the findings of a previous study by lee et al . . in general , bcs and blood glucose and bun concentrations are known to be associated with energy status and feed intake [ 7 , 8 , 38 ] . during the prepartum period , ir cows showed lower energy status and feed intake owing to the lower bcs and glucose and bun concentrations . although ir by itt was confirmed at 3 weeks before calving , a difference in energy status between ir and nir cows was noted . in particular , bcs can not be evaluated on the basis of the change in energy status of the real - time feed intake ; therefore , in this study , ir cows might have become insulin resistant during an earlier time . malnutrition causes imbalance in glucose homeostasis , and the decrease of insulin in circulation induces the reduction of feed intake in dairy cows . therefore , the feed intake reduction in ir cows might have inhibited the volatile fatty acid production in the rumen and thus suppressed gluconeogenesis in the liver . lower energy status , such as lower bcs , before calving of ir group in this study might be caused by long - term malnutrition from previous lactation . however , in this study , the reasons for the lower energy status in the ir cows were not clear ; thus , further studies are warranted to confirm the onset of insulin resistance in pregnant dairy cows . bcs at 0 and 1 week pp were lower in the ir group than in the nir group . nir cows showed higher serum nefa and bhba concentrations than those in ir cows , although the levels of metabolic hormones did not differ between the ir and nir cows . furthermore , the average and total milk yield until 100 days pp were lower in the ir group than in the nir group . higher nefa and bhba indicate greater mobilization of adipose tissue and failure of lipid metabolism in the liver [ 14 , 15 ] . however , cows with lower bcs had sustained reduced plasma nefa and bhba concentrations after calving compared to cows with higher bcs [ 28 , 33 ] . cows with lower bcs produce milk by protein mobilization , because of the limited body fat ; thus , fat - corrected milk yield in those cows was lower than moderate and fat cows . conversely , it was indicated that higher bcs cows have ability to mobilize fat to maintain energetic homeostasis after feed restriction . additionally , roche et al . have concluded that bcs at calving had positive effect on milk yield , and optimal bcs at calving was 3.5 in the 5-point scale . in the present study , greater bcs and better gluconeogenesis in nir group might produce greater milk yield compared with ir group , although the differences of them between nir and ir groups were not so greater . days to the onset of luteal activity in the ir group were fewer than in the nir group . in dairy cows , lowered energy status during the peripartum period is known to delay the first ovulation after parturition [ 2 , 19 ] . butler and smith showed that a negative energy balance was directly related to the pp interval to the first ovulation and that the differences in the energy balance were reflected in the milk yield . in addition , cows with a delayed first ovulation showed higher nefa and bhba concentrations after parturition [ 21 , 31 , 39 ] . therefore , in this study , higher nefa and bhba concentrations of nir cows during the pp period might have delayed the onset of luteal activity , and the lowered milk yield of ir cows might induce earlier resumption of ovarian activity . the maternal endocrine and metabolic milieu transferred through the placenta during late pregnancy affects the environment of the fetus [ 17 , 24 , 30 ] . in humans , ir of the mother is associated with low birth weight of the infant ; in cattle , maternal malnutrition during gestation is related to the lowered development of both the placenta and the fetus [ 24 , 30 ] . further , in ewes , restricted maternal feeding during gestation was related to lower bw and plasma igf-1 , insulin and glucose concentrations in the fetus , although maternal igf-1 concentrations were not affected . in the present study , calves of the ir cows showed lowered bw at birth and a lower plasma igf-1 concentration , supporting the findings of previous studies . in addition , they showed higher insulin levels than those of nir cows , despite the similar glucose levels . in the late gestation , fetal growth is mainly regulated by igf-1 , and the dominant regulator of igf-1 production in the fetus is fetal glucose and insulin . thus , the differences in blood metabolic hormones and glucose concentrations between the calves of the nir and ir groups might be attributed to the fetal nutritional condition that was affected by maternal endocrine and metabolic milieu . in humans , lower bw at birth is known to be associated with a wide range of adverse outcomes later in life , including diabetes ; further , obese children with low birth weight have higher blood insulin to glucose concentration and show higher insulin resistance as revealed by the homeostasis model assessment compared with obese children with normal birth weight . therefore , calves of ir cows might develop insulin resistance in the future . in conclusion , the findings of the present study suggest that ir at 3 weeks before parturition in dairy cows is related to the pp metabolic status , milk production and resumption of ovarian activity along with growth , as well as the metabolic status of their calves . therefore , ir evaluated on the basis of the recovery of glucose after an injection of a small dose of insulin during the dry period might be an indication of the pp performance of pregnant dairy cows , as well as the growth , fertility and milk production of their calves . in addition , the reason for ir in the present study was thought to be the slow recovery of glucose after insulin injection as well as the previous study . therefore , the enhancement of the gluconeogenesis in the liver by energy supplementation , such as glycerol , or hepatic stimulant , such as amino acids , should be confirmed in order to improve the ir . biodegradable block copolymers prepared from l - lactide , -caprolactone , 1,4-dioxan-2-one , and trimethylene carbonate have been synthesised and studied extensively during recent decades [ 1 , 2 ] . the varying physical properties of block polymers allows for the combination of soft and hard polymers giving rise to copolymers that can be tuned for specific function such as elasticity [ 3 , 4 ] . the sequence in which blocks are synthesised into block copolymers is specific and is determined by the choice of monomer and catalyst . for example , at the application of tin octoate as catalyst , the block copolymer structure polycaprolactone - polylactide ( pcl - pla ) is formed if the -caprolactone is polymerized first , followed by polymerization of l - lactide . however , a random copolymer is obtained when pla is initially synthesised followed by pcl [ 5 , 6 ] . this is a result of transesterification of the pla and segmentation of the block polymer . two competing reactions during carop ( coordinated anionic ring opening polymerisation ) occur ; ( i ) ring - opening of ester bonds in molecules of the initial cyclic monomer and ( ii ) cleavage of the ester bonds in the macromolecules of the polymer . these competing reactions depend on the choice of catalyst , the existing polymer , which acts as a macroinitiator , and the type of monomer added at the second stage of polymer synthesis . atering the catalyst utilized for example , y(cf3coo)3/al(iso - bu)3 catalyst or the complex [ y(l6)-{n(sihme2)2}(thf ) ] promotes the initial synthesis of the pla block , followed by the pcl block . showed that selectivity of al(oipr)3 catalyst could be accomplished through coordination with sb(oh)2 ( ( s)-()-2,20-[1,10-binaphtyl-2,20-diylbis(nitrylomethilidyne)]diphenol ) , allowing synthesis of the block structure pcl - pla - pcl , where the pla block was synthesised by the first route . furthermore , tin octoate has been shown to result in the transesterification of polyesters in the absence of monomer ; thus , the presence of the monomer initially prevents significant transesterification of the block polymer formed . catalysts possess an ability to promote transesterification reactions , and tin octoate is known for its strong ability to break ester bonds in a macromolecule . the ability to induce transesterification is a negative property of tin octoate ; however , it is commonly used in the synthesis of block polymers as the catalyst is safe to handle , inexpensive , and nontoxic . there is an interest in the synthesis of block copolymers with any given sequence of blocks using tin octoate as catalyst , though there are preconditions for this form of synthesis . it is known , that various tin - containing compounds possess different selectivity and activities in living polymerisation . it is has been shown that the activity is dependent on the valency and ligand size ; for example , tin ( iv ) displays higher catalytic activity than tin ( ii ) complexes . additionally , it has been found that the block structure pcl - pla - pcl can be obtained using tetrakis sn ( iv ) alkoxides , and it has been shown that additives ( triphenylphosphine and 4-picoline ( c6h7n ) ) can direct the carop reaction [ 11 , 12 ] . for example , it has been shown that addition of pyridine in the carop reaction promotes catalyst complexation , resulting in reduction of the competing transesterification . the propensity of tin octoate to form complexes with nucleophilic substances has been well established . have shown that the steric and electronic factors of catalysts and type of monomers utilised influence the formation of block copolymers [ 15 , 16 ] . preventing the competing transesterification reactions is an issue in the formation of multiblock polymers and is the focus of this study . through establishing an effective protocol that minimizes this side reaction a diverse range of polymers with a wider array of mechanical properties can be formed . by incorporation of a hard block in the centre of a rapidly tri- or starblock structure commonly , blocks possessing high degradability that terminate the macromolecule lead to a high rate of degradability for the entire copolymer and vice versa . furthermore , the hard block core surrounded by soft bloeks with low degradability can furnish tough biodegradable copolymers with slow degradation rates . these features are desirable properties in the production of biodegradable packaging that are expected to have long enough shelf life time . additionally , when crosslinked , block structures with flanking hydrophobic regions allow the formation of hydrogels with high swelling capacity , a feature required in scaffold - based tissue engineering . for ring opening polymerization to remain as a living polymerization , preventing transesterification and preserving the lengths of blocks polymers formed is important and allows for the synthesis of multiblock polymers with controlled repetition of hard and soft blocks . additionally , these improvements would allow incorporation of carop reaction into the field of biomimetics whereby the properties of naturally occurring materials could be mimicked such as the adhesive materials of sea shells or spiders silks . such polymer structures with a hard block in the middle or a highly repetitive sequence of hard and soft blocks can be useful in the production of stents and occluders such that the mechanical properties and degradation behavior can be mimicked . hence , we investigated the optimisation of carop reaction to maintain integrity of the 1st pla block in block copolymer formation . within this study , we investigate three possible variants in polymerisation utilising tin octoate to improve the carop reaction and minimise transesterification side - reactions , ( i ) identify a suitable protective additive , thereby protecting the pla formed from competing side - reactions , ( ii ) identify an additive that minimises transesterification by mimicking the characteristics of the initial monomer , and ( iii ) identify a suitable additive that is able to complex to tin octoate thereby minimising the competing transesterification reaction while promoting the cycle - opening reaction for the monomer . -caprolactone ( 99% purity ) , obtained from fluka , was dried over cah2 and distilled under nitrogen at reduced pressure . ( 3s)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione ( l - lactide ) ( 98% purity ) was obtained from sigma aldrich and purified by recrystallization with dry diethyl ether . 1,3-trimethylene carbonate ( 99% purity ) from boehringer ingelheim corporation , germany , was used without further purification . the monomer was dried for 24 h under reduced pressure at room temperature prior to polymerization . tin 2-ethylhexanoate ( 96% purity ) ( sn(oct)2 ) from sigma aldrich , hydroxy butyl vinyl ether ( 98% purity , hbve ) stabilized by 0.01% koh from basf , and 1,4-butanediol ( 99% purity ) from alfa aesar were purified by distillation under nitrogen at reduced pressure . methanol anhydrous ( 99.8% purity ) from sigma aldrich and tetrahydrofuran ( 99% purity ) from alfa aesar were used without further purification . toluene anhydrous ( 99.8% purity ) from sigma aldrich was dried over cah2 and distilled under nitrogen . synthesis was performed in a three - necked round bottom flask ( 100 ml ) equipped with a thermometer , a condenser and a magnetic stirrer . the flask was purged with dry argon and vacuumed twice , after which the reaction vessel was kept under the argon atmosphere . toluene , 1,4-butanediol or hbve ( initiators ) , and sn(oct)2 ( catalyst ) were added to the flask at 90c and stirred for 30 minutes . the quantity of the initiator and monomers used were based on the desired degree of polymerization . the quantity of the catalyst sn(oct)2 was chosen such that the ratio of initiator to catalyst was maintained at a constant of 10 for all syntheses . the necessary quantity of monomers ( -caprolactone or l - lactide or -caprolactone and l - lactide ) was added to synthesize the first block of copolymer , depending on desired polymer structure . the temperature was then increased to 110c for 24 hours for polymerization to proceed . to synthesize the 2nd block , the necessary quantity of monomers ( -caprolactone or l - lactide or trimethylencarbonate ) was added to the flask and allowed to react for a further 24 hours at 110c . mol / l , and the quantity of monomers usually taken for the synthesis was 0.08 mol . after 48 hours , the reaction mixture was poured into cold methanol and the precipitated polymer was filtered , washed several times by cold methanol , and dried in a vacuum oven for 48 hours at 40c . the molar masses of polymers were determined by size exclusion chromatography ( sec ) using an agilent 1100 series hplc . polystyrene standards with a narrow molar mass distribution in the range of 580400,000 g / mol were used for calibration . measurements were made at room temperature with a linear pl gel and 5 m mixed c column . chloroform was used as eluent with a flow rate of 1 ml / min . samples were prepared in deuterated chloroform ( 200 mg of polymer/1 ml cdcl3 ) . c - nmr and h - nmr spectra were obtained using a bruker 400 spectrometer with deuterated chloroform used as internal standard . average lengths of poly(-caprolactone ) ( lcl ) and poly(l - lactide ) blocks ( lla ) were calculated from the intensities of the carbonyl signals [ 23 , 24 ] using the equations below : ( 1)lpcl = icccilcc+1,lpla = illlilllc+1 , where iccc and illl are the intensities of -caprolactone--caprolactone and l - lactide - l - lactide triads , respectively ; ilcc and illlc represent the intensities of -caprolactone - l - lactide and l - lactide--caprolactone triads and tetrads peaks , respectively . the glass transition temperatures and melting enthalpies of polymer samples were measured using a ta instruments model q10 dsc machine equipped with a dsc refrigerated cooling system and ta instruments control software . dsc analysis was accomplished by initially heating the sample to 200c to eliminate internal stresses . samples were then equilibrated at 80c , followed by heating samples to 200c at a rate of 10c per minute . crystallinity ( c ) of the polymers was calculated using ( 2 ) : ( 2)c=hh100100 , where h is the experimental melting enthalpy of polymer in j / g and h100 is the melting enthalpy of the polymer with 100% crystallinity and h100 = 139 j / g for polycaprolactone and h100 = 93 j / g for polylactide . mass spectrometric measurements were performed using a kratos axima tof ( kratos - shimadzu biotech , manchester , uk ) time of flight instrument , equipped with a pulsed n2 laser ( 337 nm , 4 ns pulse width ) and time - delayed extraction ion source . the matrix , 2,5-dihydroxybenzoic acid ( dhb ) or dithranol , was dissolved in thf ( 20 mg / ml ) . sodium iodide was dissolved in thf ( 5 mg / ml ) and used as the ionizing agent . samples were prepared by mixing the matrix solution with the polymer solution and ionizing agent to the ratio of 10 : 1 : 1 , respectively . this mixture ( 1 l ) the average molar mass of polymers was calculated using the standard software program provided by the instrument manufacturer . gas chromatography - mass spectrometry measurement was recorded by quattro micro gc with quadrupole mass spectrometer ( waters corporation ) . the length of pla - pcl block copolymer formation can be influenced by additives that prevent segmentation due to tin octoate . linear , branched saturated or unsaturated esters , styrene and -methylstyrene were considered as suitable additives to minimise transesterification side reactions induced by tin octoate . initially , protective additives ( 10 : 1 initial monomer : protective additive ) were added to reactions upon synthesis of the 1st copolymer block . for all reactions , the targeted triblock pcl - pla - pcl was 20 - 40 - 20 kda and the physical characteristics of the obtained copolymers are outlined in table 1 . analysis of the nmr spectra showed that block copolymers where obtained in the presence of protective additives -methylstyrene , styrene , and ethyl benzoate . the presence of the protective additive esters was found to preserve the pla macromolecule , providing longer pla and pcl segments in comparison to reaction where no additive was utilised . the triblock molar mass closest to the required target was achieved when -methylstyrene was utilised . furthermore , an increase in the molar mass of the polymer was observed during generation of the 2nd copolymer block with living polymerisation sustained . 2 ) showed the observed increments of polymer molar mass by analysing the middle pla block , initially synthesised , and the final triblock copolymer by sec ( figure 1 ) . to study the influence of protective additives in the formation of copolymers , a series of reactions were performed neat with the chosen additives . upon treatment of the monomers and tin octoate , protective additives , -methylstyrene , and styrene yielded pcl - pla - pcl block copolymers with high crystallinity of pcl and pla . furthermore , introduction of the protective additive promoted the preservation of the block structure and augmentation of the pcl and pla segment lengths . from the aforementioned data ( table 1 ) , increased polymer lengths were observed when additives containing electron - rich motifs such as the saturated olefin observed in -methylstyrene were utilised . additionally , -methylstyrene is known to homopolymerize only at low temperature ( 78c ) through an anionic mechanism , due to its low ceiling temperature ( 66c ) compared to styrene ( 395c ) . since reactions of cyclic monomers was performed at 110c , -methylstyrene was further investigated in improving the polymerisation reaction as it would not interfere with the block copolymer formation . initially , a range of molar ratio of protector additive and tin octoate was investigated for carop reactions or cyclic monomers . the targeted molar mass of the triblock pcl - pla - pcl was 20 - 40 - 20 kda for all experiments . the obtained molar masses and crystallinity for each synthesized polymer are summarized in table 2 . varying the ratio of -methylstyrene compared to the catalyst from 2.5 : 1 to 2500 : 1 resulted in improved polymerisation reactions as the additive ratio increased ; however , the increase in -methylstyrene significantly reduced the rate of ring opening polymerization . when the protective additive : catalyst ratio was higher than 125 , a decrease in the resulting molar mass of the polymer obtained was observed . additionally , polymer 3 ( table 2 ) displayed the lowest pla crystallinity , while the pcl segment displayed no crystallinity . the molar ratios of monomers l - lactide and -caprolactone used and the resulting segment lengths of the copolymers are outlined in table 3 . c nmr analysis at 160180 ppm of the obtained polymers from table 3 demonstrated the formation of the desired copolymers , figure 2 . evidently , analysis of the crystallinity ( table 2 ) , block lengths ( table 3 ) , and nmr data ( figure 2 ) polymer 3 ( table 2 ) does not conform to the observed trend . the presence of dyads and triads as observed in polymer 6 ( figure 2(d ) ) suggests that polymer 3 ( figure 2(b ) ) also exists as a random copolymer due to the transesterification side reactions . to elucidate the mode of reaction for polymer 3 , we reacted tin octoate with -methylstyrene in toluene without a cyclic monomer ; however , gc - ms and maldi - tof analysis ( section 3.3 ) did not elude to the phenomena observed with polymer 3 ( table 3 ) . comparing of pcl and pla blocks length of polymers 4 and 5 ( table 3 ) with polymers 13 , we speculate that another mechanism of protection occurs when the quantity of protective additive is significantly higher . polymers 4 and 5 ( table 3 ) exhibit the largest pcl and pla segment lengths and lowest molar masses . comparing the influence of styrene and -methylstyrene , we propose that the mechanism of segment protection with -methylstyrene is a result of its higher propensity to participate in nucleophilic substitution reactions compared to styrene . it is possible that the slight difference in structure between -methylstyrene and styrene is important as it is known that the difference in one methyl group in a ligand for a given catalyst can influence the resulting product of the carop reaction . analysing the aforementioned data , a nonlinear dependence of differing quantities of -methylstyrene can be deduced and it appears that -methylstyrene interacts only with catalyst and does not react with the monomer ( -caprolactone ) or with polymer - macroinitiator ( pla ) . a series of model reactions structured on the synthesis of polymer 3 ( random structure ) were performed to determine the mode of reaction for -methylstyrene : ( i ) a twofold increases in the monomer quantity , ( ii ) twofold increase in initiator ( 1,4-butanediol ) , ( iii ) fourfold reduction in catalyst ( tin octoate ) , and ( iv ) twofold dilution ( toluene ) of the reaction mixture . it was found with increases in monomer quantity ( reaction 1 ) and initiator ( reaction 2 ) , random copolymers were obtained as observed for polymer 3 ( tables 2 and 3 ) . reduction of tin octoate ( reaction 3 ) leads to the formation of a block copolymer with pcl crystallinity of 44.4% and pla crystallinity of 45.3% , while dilution of the reaction solution gave a polymer with pcl crystallinity of 9.2% and pla crystallinity of 39.2% . the obtained pcl and pla segment lengths of copolymer ( reaction 3 ) were 22.2 and 32.9 monomer units , respectively . thus it can conclude that the protective additive ( -methylstyrene ) reacts only with the catalyst ( tin octoate ) , and the concentration of the reacting substances influences the degree of interaction between the catalyst and the protective additive used thereby dictating its effectiveness . to determine the products of the reaction between only -methylstyrene and tin octoate , three experiments with differing molar ratios of tin octoate : -methylstyrene were conducted . the molar ratios ( tin octoate : -methylstyrene ) utilised were ( i ) 1 : 2.5 , ( ii ) 1 : 125 , and ( iii ) 1 : 300 . maldi - tof analysis of the resulting products was utilised and potential products of reactions are outlined in table 4 . the most intensive signals on maldi - tof spectra were obtained for structures 3 , 4 , 6 , 7 , 8 , and 10 ( table 4 ) . interestingly the most intense signal was due to substance 6 ( table 4 ) with a mass of 413.33 da , which was observed in all three model reactions . due to the diversity of the products obtained in table 4 , we could not conclude through maldi - tof analysis the compounds responsible for random copolymer formation . hence , we performed syntheses of triblock copolymers of pcl - pla - pcl using the three molar ratios of protective additive : tin octoate . the targeted molar mass of the copolymers for maldi - tof analysis was 2000 - 2000 - 2000 da ( pcl - pla - pcl ) . analysis of the maldi - tof spectra showed the molar masses of polymers synthesised with tin octoate : -methylstyrene ratios of 1 : 2.5 ( 5130 da ) , 1 : 125 ( 4010 da ) , and 1 : 300 ( 4640 da ) . the ratios of l - lactide : -caprolactone for these copolymers , determined from h nmr , were 43 : 57 , 42 : 58 , and 44 : 56 , respectively . maldi - tof analysis of the copolymers formed ( figure 3 ) displayed a difference of 72 da , indicative of one monomer unit of lactic acid suggesting that the polymers underwent transesterification . spectrum ( b ) ( figure 3 ) displayed additional signal compared to spectra ( a ) and ( c ) indicating that an increased degree of transesterification had occurred . all significant peaks acquired from maldi - tof spectra were used in determination of the end groups . from the literature , we considered published data regarding the analysis of terminal groups of similar copolymers [ 2628 ] . analysing the monomer ratio determined through nmr and mass spectra obtained from maldi - tof analysis , copolymer synthesised with a tin octoate : -methylstyrene ratio of 1 : 2.5 contained the following terminal groups in abundance with molar masses : 188 , 385 , 397 , 413 da . the tin octoate : -methylstyrene ratio 1 : 125 formed the following terminal groups : 260 , 368 , 394 , 385 , and 397 da . the tin octoate : -methylstyrene ratio of 1 : 300 resulted in the formation of terminal groups 117 , 172 , 188 , and 260 da . it is probable that the interaction between the catalyst and -methylstyrene forms a wide spectrum of products that depend on the ratio of the reacting components . we attempted to explain the observed differences of copolymers from analysis of the maldi - tof spectra . at equal ratios of catalyst : -methylstyrene , the electrophilic attack of electrophilic sites of tin octoate ( between carbonyl carbon and tin atom ) by -methylstyrene is possible . the coordination of tin octoate by -methylstyrene occurs , as maldi - tof spectra displayed the required mass ions of 521 , 522 , and 523 da ( tin octoate + -methyl styrene h 405.12 + 118.18 1 = 522.3 da ) . studies have shown the capability of tin octoate to react with aibn , producing a complex with better catalytic working capacity . additionally , steric factors are important as tin ( iv ) does not participate in complex formation at similar conditions . we expect that complex molecules containing tin ( substances 28 from table 4 ) are formed . thus , by altering the steric structure of the catalyst used transesterification can be minimised , thus conserving the pla blocks that are formed first during polymerisation . from calculations and our experiences with these substances , compound 6 from table 4 , ms co o sn o - ms , with a mass of 413 da is most likely responsible for the protection of pla at the catalyst : -methylstyrene ratio of 1 : 2.5 . with increases of the tin octoate : -methylstyrene ratios up to 1 : 100 , the probability of interactions between the electrophilic sites of tin octoate and -methylstyrene increases . it is likely that the products from these reactions become less branched and more symmetrically linear . we speculate that the transformation of tin ( ii ) into tin ( iv ) possibly occurs . thus , the catalytic activity of the transformed catalyst increases in comparison to the initial tin octoate . a series of tin ( iv ) alkoxides have been shown to be the most active catalysts for carop . in our examples , an increase in the observed mass at 260 ( compound 13 , table 4 ) , ms o co r , suggests that at these reaction conditions , the concentration of -methylstyrene becomes sufficient to destroy two branches of tin octoate , giving different compounds . furthermore , the number of tin atoms coordinated by several molecules of -methylstyrene ( up to four ) increases among the products of reaction . from analysis of the maldi - tof and nmr spectra , and consideration of the random copolymer formation with a catalyst : -methylstyrene ratio of 1 : 125 , we can assume that this is caused by the appearance of substances 3 , 7 , and 8 ( table 4 ) in the reaction medium . finally , considering the terminal groups of the macromolecules , we suspect that the increase of transesterification activity was induced by symmetric compound 8 from table 4 with mass 385 da , ms o sn o ms . this has been shown by majerska et al . in which the formation of substances with similar structure , sn(or)2 , using tin octoate have been established and confirmed . with the further increase in the concentration of -methylstyrene , complete destruction of tin octoate is possible , and domination of tin atoms coordinated by molecules of -methylstyrene starts to prevail . catalytic activity is maintained , though it is considerably reduced in comparison with that of tin octoate owing to the massive structure and shielding of tin by ligands . it has been well proved by the incomplete reaction of monomer and low - molar - mass polymer synthesised at a catalyst : -methylstyrene ratio of 1 : 300 and above . moreover , the transesterification ability of tin vanishes for the same reason tin is shielded by aromatic rings of -methylstyrene . there is also an increase in the content of small fragments born from tin octoate that can react with 1,4-butanediol and -methylstyrene ( substances 1214 from table 4 ) . to further probe this reaction , we reacted -methylstyrene and tin ( ii ) octoate , in different ratios at reflux in toluene for 24 h and subsequently analyzed the obtained products . gc - ms analysis of the mixture revealed the presence of intermediate benzeneacetic acid-2-ethylhexyl ester ( scheme 1 ) , due to the interaction between -methylstyrene and tin ( ii ) octoate . benzeneacetic acid-2-ethylhexyl ester ( scheme 1 ) was observed as the major compound in all varying ratios . additionally , several intermediates originated from tin ( ii ) octoate were also identified , including 2-ethyl hexanoic acid , 2-ethylhexyl 2-ethylhexanoate , and hexanoic acid 2-ethyl anhydride . therefore , it was confirmed that the mechanism of the -methylstyrene protection is associated with -methylstyrene and tin ( ii ) octoate complexation . the protective properties of -methylstyrene were tested in the synthesis of diblock structures pla - pcl ( pla first route ) and triblock copolymer ptmc - pla - ptmc . we synthesised a reverse triblock ptmc - pla - ptmc in toluene using tin octoate producing a random copolymer . we obtained a ptmc - pla - ptmc triblock copolymer using -methylstyrene : tin octoate ( 125 : 1 ) , through a two - step reaction in toluene . the targeted structure of copolymer ptmc - pla - ptmc was 20 - 40 - 20 kda . the obtained properties of the block copolymers including the molar masses , crystallinity , and segment lengths of pla , and l - lactide : trimethylencarbonate ratio for two copolymers are presented in table 5 . it can be seen from figure 4 , that the use of protective additive resulted in no signals in the region of 120140 ppm ( spectrum b ) indicating that the transesterification processes did not occur . the dyad signal from the carbonyl of ptmc ( 154 ppm ) is observed on both spectra , while the dyad signal from l - lactide ( 169 ppm ) is seen only in the spectrum ( b ) , hence confirming the formation of the block structure . furthermore , the spectrum ( a ) ( reaction without protective additive ) displays smaller carbonyl peaks , indicating that reaction without the presence of the protective additive results in increased formation of random polymers . we then utilised -methylstyrene as protective additive for the synthesis of the diblock pla - pcl ( pla first rout ) . all reaction conditions were performed as previously stated with a molar ratio of tin octoate : -methylstyrene ratio of 1 : 25 ; however , hbve was used as polymerisation initiator . the targeted molar mass of diblock copolymer pla - pcl was 40 - 40 kda , which upon reaction was successfully furnished . c nmr spectra of pla - pcl displayed two carbonyl peaks in the region 160170 ppm resulting from the formation of the desired diblock , figure 5 . we note that the nmr spectra of both the tri - block ( figure 2 ) and di - block ( figure 5 ) copolymers display minimal difference in signals observed . this is a result of polymer fabrication , whereby in both examples the pla block is formed first followed by the pcl block . in both examples , we can observe in the nmr spectra the absence of carbonyl signals due to triads , tetrads , and randomised segments . as highlighted , the addition of a small quantity of -methylstyrene can preserve the pla macromolecules polymerised during the initial sequence of block copolymer synthesis ( pcl - pla - pla or pla - pcl ) . however , it is difficult to reach molar masses greater than 6070 kda for these polymers using the carop reaction due to transesterification [ 33 , 34 ] . we attempted the synthesis of pcl with a target molar mass of 100 kda using the reaction conditions previously outline with protective additive -methylstyrene ( tin octoate : -methylstyrene molar ratio ; 1 : 25 ) and without . sec analysis of the reaction products showed an mn 108.1 kda for pcl synthesised with -methylstyrene , while an mn of 72.3 kda for pcl was observed when no protector additive was used , figure 6 . we conclude that the addition of protective additives decreases the transesterification side - reactions that are especially important to the synthesis of polymers with high molar masses . it is known that after 9599% of monomer conversion , polydispersity of the polymer increases as the rate of transesterification side - reaction begins to increase at minimal quantities of monomers . we investigated this phenomena and attempted to control polydispersity through addition of the protective additive -methylstyrene . pla ( 5 kda ) was utilised as polymer ( a ) for investigation , as shown in the top spectrum ( figure 7 ) . reactions of ( i ) tin ( ii ) octoate in the presence of -methylstyrene ( b ) and ( ii ) only tin ( ii ) octoate ( c ) with starting polymer pla were investigated . model reactions were performed by dissolving 5 g of pla , 40 l of sn(oct)2 , and 40 l of -methylstyrene in 50 ml of toluene and allowing the mixture to heat to refluxing under argon atmosphere for 24 hours . analysis of the maldi - tof spectra of the initial pla and the two pla samples from the pilot studies highlighting the polydispersity are outlined in figure 7 . molecular mass ( mn ) of the obtained pla samples ( figure 7 ; ( a ) , ( b ) , ( c ) ) and the polydispersity index were determined utilising software to give an mn of 4915 da ( a ) , 4415 da ( b ) , 3414 da ( c ) and a polydispersity of 1.20 ( a ) , 1.22 ( b ) , and 1.41 ( c ) . evidently , the introduction of -methylstyrene reduced the negative effect of transesterification induced by tin octoate and decreased the polydispersity . this study has established that protective additives -methylstyrene and styrene are capable of reducing the influence of the transesterification side - reactions during the synthesis of block copolymers of polycaprolactone and polylactide or polytrimethylenecarbonate and polylactide . mechanistic studies can not elude to the mode that these protective additives suppress transesterification as there is no linear dependence of transesterification versus tin octoate : -methylstyrene molar ratio . it can be suggested that the formation of products in the carop reaction is dependent on the ratio of tin octoate : -methylstyrene . ms , with mass 413 da obtained during treatment of tin octoate with -methylstyrene is responsible for the reduction in transesterification and protection of the block polymers . the effectiveness of this model of polymer protection was tested with successful formation of triblock ( ptmc - pla - ptmc ) and diblock ( pla - pcl ) structures . the ability of -methylstyrene to decrease the transesterification reaction was also analysed by refluxing pla with an amount of tin octoate in toluene . the polymer treated with the addition of -methylstyrene remained almost unchanged while without the protective additive significant transesterification occurred . in conclusion , the application of protective additives in ring opening polymerisation will allow for the formation of block copolymers with consistent uniform structures and high molecular mass as well as the generation of previously unobtainable polymers with new physical features and properties . ultimately this new protocol could be applied to the field of biomimetics , allowing the synthesis of novel biodegradable materials that could be used in the medical industry ( as stent , occluders , scaffold tissue engineering , and suture ) and manufacturing industry ( as packaging material ) .

this study aimed to investigate the effects of insulin resistance ( ir ) during the close - up dry period on the metabolic status and performance of dairy cows as well as to determine the effects on body weight ( bw ) and metabolic status of their calves . an insulin tolerance test ( itt ) was conducted by administering 0.05 iu / kg bw of insulin to 34 multiparous holstein cows at 3 weeks prepartum . blood samples were collected at 0 , 30 , 45 and 60 min after insulin injection , and cows were divided into two groups based on the time required for glucose to reach the minimum levels [ non - ir ( nir ) , 45 min ( n=28 ) ; and ir , 60 min ( n=6 ) ] . blood or milk sampling and body condition score ( bcs ) estimation were performed twice weekly during the experimental period . blood samples from calves were collected immediately after birth . cows with ir showed lower bcs ( p<0.05 ) and serum urea nitrogen ( p<0.05 ) and glucose concentration ( p=0.05 ) before calving , and lower serum non - esterified fatty acid concentration ( p<0.05 ) and milk yield ( p<0.05 ) and earlier resumption of luteal activity ( p<0.05 ) after calving ; their calves showed lower bw ( p<0.05 ) and plasma insulin - like growth factor - i concentration ( p<0.001 ) and higher plasma insulin concentration ( p<0.05 ) . in conclusion , ir at 3 weeks prepartum in dairy cows is related to postpartum metabolic status and performance along with growth and metabolic status of their calves . well - defined di- and triblock copolymers consisting of -caprolactone ( cl ) , l - lactide ( la ) , and trimethylene carbonate ( tmc ) were synthesized via pla first route in coordinated anionic ring opening polymerization / copolymerization ( carop ) with tin ( ii ) octoate as catalyst . the desired block structure was preserved by use of protective additive -methylstyrene by preventing the transesterification side - reactions . maldi - tof analysis revealed that the protection mechanism is associated with -methylstyrene and tin ( ii ) octoate complexation . additionally , it was shown that use of -methylstyrene in ring opening polymerization allowed the formation of polyesters with high molar mass .

the rapid improvement in quality , quantity , and cost of next generation sequencing ( ngs ) has resulted in commensurate improvements in analysis techniques . for bacteria , the availability of kits for library preparation , rapid and high content sequencing , and mature data analysis pipelines for genome resequencing and assembly had drastically reduced costs and improved reliability of these results . the commoditization of bacterial genome sequencing has led to more complex applications : clinical and agricultural diagnostics [ 14 ] , outbreak detection and monitoring [ 57 ] , human health studies [ 8 , 9 ] , biocatalysis [ 10 , 11 ] , environmental studies , and many others [ 13 , 14 ] . for ngs platforms , current sequencing technologies require that sequencing adapters be ligated to dna fragments before sequencing is possible . ligation of adapters to ( typically small ) dna fragments is an inefficient process , generating ligated hybrids from only a small fraction of targeted dna molecules . this limitation in turn increases the required dna input , with the only goal being to generate sufficient numbers of ligated fragments to allow sequencing . typical library preparation methods require large amounts ( ~1 g ) at high concentrations ( > 25 ng / ml ) of dna for successful library generation , limiting the types of samples that can be sequenced reliably . these include high variability of evenness and completeness of genome coverage as a function of % gc content , input dna quantities , and sequencing technology [ 1519 ] . these impact the amount of sequencing data required and the quality of genome assembly and analysis . several library preparation kits that require 1100 ng of input dna are now available ( new england biolabs ' nebnext , illumina 's truseq nano , bioo scientific 's nextflex , nugen 's ovation ultralow , etc . ) . this paper details the results of evaluation of the utility of the nebnext ultra library preparation kits for both resequencing and assembly of several bacterial genomes . we compare the evenness and completeness of coverage between nebnext ultra and illumina truseq kits for bacterial genomes of varying size and % gc content . our findings indicate that low dna input amounts are sufficient to generate high quality sequencing data that can be used for genome resequencing or de novo assembly ( if combined with long fragment data ) . we sequenced three different bacterial species with various genome lengths ( from 5.4 mb to 6.7 mb ) and containing various % gc contents ( from 35% to 68% ) . standard input dna amounts were 100 ng , approximately 10x lower than the required amount for the illumina truseq kit and 10x higher than the minimum dna inputs per nebnext ultra manual specifications . the most challenging ( longest genome and highest gc content ) bacterial genomes ( burkholderia a and b ) were also sequenced with minimal dna inputs ( 10 ng ) . all samples were sequenced on the illumina hiseq platform using 2 100 bp chemistry . data analyses consisted of read - mapping the short fragment data to reference genomes using bwa ( burrows - wheeler alignment ) . these data were also combined with long insert mate pair data to evaluate their utility for de novo assembly of the bacterial genomes . genomic dna from bacillus anthracis ( strain sterne 34f2 ) was isolated from a log phase culture using the mo - bio ultraclean microbial dna isolation kit . burkholderia thailandensis a ( strain e254 , accession numbers cp004381 and cp004382 ) and burkholderia thailandensis b ( strain usamru malaysia # 20 , accession numbers cp004383 and cp004384 ) are previously reported strains , and dna was provided by dr . the integrity of all genomic dna samples was evaluated using agarose gels and their quantity measured with picogreen reagents on a qubit 2.0 instrument . nebnext ultra library preparation protocol consists of several enzymatic and two purification steps , one of which is used for size selection of library fragments . genomic dna samples were sheared in 55 l of tle buffer ( 10 mm tris , 0.1 mm edta , ph 8) using covaris e220 with the following settings : duty cycle 10% , intensity 5 , cycle 200 , and time 100 sec . after shearing , two enzymatic steps ( end preparation and adapter ligation ) are performed in the same tube , followed by size selection of the library fragments using a double ampure cleanup . first ampure step used 0.4x sample volume of beads and the supernatant was transferred to a clean tube . selected library fragments were amplified with barcoded primers ( 1012 pcr cycles ) and purified one more time with ampure beads ( 0.5x bead volume ) ( see supplementary material available online at http://dx.doi.org/10.1155/2014/434575 ) . nebnext libraries were analyzed using bioanalyzer 2100 and dna 1000 or dna high sensitivity chips , to quantify the library size and assess the level of adapter - dimer and primer - dimer contamination . libraries were quantified using illumina library qpcr quantification kits from kapa biosystems and sequenced on either the illumina miseq or illumina hiseq . the illumina data from this study were trimmed to remove any ambiguous bases ; any reads shorter than 70 bp after trimming and the corresponding read pairs were discarded . the total number of reads per sample ranged from 6.2 million to 47.8 million before trimming . all data had read lengths of 151 bp with one exception which had read lengths of 101 bp . after trimming , the average read lengths were reduced by less than 3.5% for all samples . the data for each sample were normalized to 70x coverage of the genome after trimming . the average number of reads with a quality greater than q20 after trimming and normalization ranged from 61% of the total reads to 97% of the total reads . the total number of reads , the number of reads with quality greater than q20 , and the average read lengths before and after trimming for each sample can be found in table s1 . the assemblies were compared to the reference genomes to consider insertion / deletion errors and rearrangements using an in - house perl script . for read - mapping , burrows - wheeler alignment ( bwa ) mapping tool was used , combined with samtools and in - house perl scripts for coverage and insert size analysis [ 21 , 22 ] . for base coverage we used bwa global alignment option with default parameters . bwa global alignment only reports the best alignment based on score calculated by a set of parameters . if a read has several possible best alignment spots , bwa randomly assigns the read to one spot . all reads mapped to contigs were used to calculate base coverage . for insert size calculation , only properly paired reads ( read pair on the same contig and with correct orientation ) were used . we report the mean , standard deviation , the minimum , and maximum of the insert size distribution for all short fragment libraries . we utilized three thresholds for reporting coverage : 0% , 1% , and 10% of mean fold coverage . calculation of evenness of coverage was performed by calculating the average and standard deviation of coverage across nonoverlapping 10 kbp fragments of the finished genome . evenness for each fragment was calculated as 1 ( standard deviation of coverage / coverage ) . all data points ( genomic and plasmid coverage , where appropriate ) were used to generate box and whisker plots in ibm 's statistics program spss . two debruijn graph assembly tools were used to evaluate the quality of the short fragment data for the purpose of assembling high quality genomes . paired reads were randomly selected ( in silico ) from each sample to generate libraries of approximately 70-fold genome coverage for each sample , in order to normalize the data . the only exception was the e. coli sample prepared with the truseq kit , for which only 61-fold coverage was available . the 70-fold short fragment illumina data were combined with previously sequenced long insert mate pair data generated by 454 . the 454 data had an average insert size of 8 kbps and provided 7- to 8-fold base coverage , with the exception again for the e. coli samples , which had approximately 3.5-fold coverage . the library preparation protocol , as described in section 2 , yields average insert sizes of ~270 15 bps ( average library sizes of ~400 15 bps ) that are optimal for either 2 100 or 2 150 bp sequencing on illumina platforms . different insert sizes can easily be obtained by adjusting the size selection step ( ratio of dna solution to ampure beads ) as recommended by the manufacturer . it is not necessary to adjust the shearing step , as the sheared dna produced by covaris has a very broad size distribution . nebnext library process provides very consistent results in terms of library size and concentration , even when performed for the very first time . prior to normalization and sequencing , samples were analyzed using qubit ( picogreen - based method ) , bioanalyzer 2100 , and quantitative real - time pcr ( qpcr , kapa biosystems ) . unfortunately , this was not the case when molar library concentrations were obtained with qubit and bioanalyzer data only ( without qpcr ) . sequencing was performed on either illumina hiseq ( 2 100 bp ) or illumina miseq ( 2 150 bp ) . figure 1 ( b. anthracis and e. coli ) and figure 2 ( b. thailandensis a and b ) contain sliding window coverage plots that compare the coverage of each genome by different library preparation method and different dna input amount . from the figures , it can be seen that the genome coverage is remarkably similar regardless of the library preparation method ( nebnext or truseq ) . of particular interest is that even the libraries prepared from only 10 ng of genomic dna produced essentially the same evenness of genome coverage as the rest of the samples ( figures 2(a)2(d ) , top panel ) . as table 1 shows , the number of true gaps in coverage ( 0% ) is slightly higher for nebnext than for truseq libraries , while the number of gaps is lower for nebnext when using 1% or 10% average coverage thresholds . the data were not normalized among all samples prior to evenness of coverage comparisons . instead , they were normalized within each sample relative to the average coverage . figure 3 shows box and whisker plots of the evenness of coverage of 10 kbp windows for each genome . in the case of e. coli , the evenness of coverage for nebnext ultra libraries prepared with 100 ng of input dna is superior to that of truseq libraries produced with 1 g of input dna . for b. anthracis and b. thailandensis , there is more variation in coverage for the nebnext preparations at both 100 ng and 10 ng . further examination of this effect suggests that it is proportional to the amount of input dna , supporting the theory that nebnext ultra kits either do not introduce bias or introduce similar bias to truseq kits , with a lower dna requirement . figure 4 shows the results of de novo genome assemblies generated using the short fragment data either alone ( figure 4(a ) , assembled with idba ) or complemented with long insert mate pair data ( figure 4(b ) , assembled with allpaths ) . idba assemblies show very similar results for low ( b. anthracis ) to medium ( e. coli ) % gc genomes . however , data obtained from nebnext libraries show a dramatic reduction in the number of contigs for high % gc genomes from the two burkholderia strains . importantly , nebnext libraries prepared from just 10 ng of genomic dna maintain the high quality of genome assembly , producing similar numbers of contigs as with 100 ng input dna samples . however , scaffolding mostly depends on the long insert mate pair data , which are the same for all assemblies . comprehensive assembly statistics are shown in tables s2a and s2b . in conclusion , we have demonstrated that the quality of bacterial genome resequencing and de novo assembly is similar , regardless of the library preparation method and input dna amount ( from 10 to 1000 ng ) . the only significant difference was observed in the assemblies of the b. thailandensis genomes , where nebnext library data produced dramatically more contiguous assemblies ( figure 4 ) . in general , the assemblies from the truseq libraries were more prone to indels and rearrangements . the full results for the comparisons of our assemblies to the reference genomes are found in table 2 . this is likely due to the improved ability of the nebnext reagents to more effectively amplify high % gc regions that are very common in burkholderia genomes . modern library preparation methods for next generation sequencing technologies , as represented by nebnext ultra , are enabling bacterial genome sequencing from very small input amounts of genomic dna . these methods likely do not require any additional improvement , since handling and quantifying dna amounts smaller than 10 ng can become challenging . we have deposited the genomes of burkholderia thailandensis a ( strain e254 , accession numbers cp004381 and cp004382 ) and burkholderia thailandensis b ( strain usamru malaysia # 20 , accession numbers cp004383 and cp004384 ) into genbank . all experimental and computational methods used during this work are publically available or can be provided by the authors . lateral epicondylitis is also known as tennis elbow or tendonitis of the extensor muscles of the forearm and refers to pain and tenderness over the lateral epicondyle of the humerus ; this pain is exaggerated by resisted dorsiflexion of the wrist or the middle finger . it has been estimated that 1 - 3% of the population suffer from this condition with equal distribution between men and women . pathophysiology of lateral epicondylitis most commonly involves osteotendinous part of the extensor muscles of the wrist at their origin ( the lateral epicondyle ) ; among these , the tendon of the extensor carpi radialis brevis is more commonly involved . it is believed that the injury involves tears ( either microscopic or macroscopic ) in the origin of the extensor muscles of the wrist , which leads to an inflammatory response and in the chronic cases granulation and fibrous tissue . studies have shown that most cases of lateral epicondylitis involve fibrotic tissue and angiogenesis as a result of which some believe that tendinosis is a more correct term for the condition than tendinitis . it has been argued that the most common causes of lateral epicondylitis are overuse injuries . tendons are relatively hypovascular and as a result are prone to injuries which are cause by hypoxia such injuries usually result from occupational and athletic activities . these activities include ( but are not limited to ) : backhand stroke in racquet sports , pitching in baseball , typing on keyboards , repetitive occupational hand movements such as hammering or driving screws and carrying heavy briefcases . symptoms of the condition include pain on the lateral aspect of the elbow , reduced grip , increase in pain with activity and reduced strength of the extensor activities of the wrist . despite the fact that this condition often lacks an inflammatory component the main sign of the lateral epicondylitis is tenderness of the lateral epicondyle coupled with pain on resisted dorsiflexion of either wrist or middle finger . the evidence shows that 95% of patients heal either spontaneously or by conservative measures alone . this has led some practitioners to believe that it is not necessary to treat lateral epicondylitis , their argument is supported by studies such as the study by smidt et al . which showed a limited benefit for treatment modalities other than expectant management . one of the treatments which has unequivocal evidence both for and against it , is corticosteroid injection . a meta - analysis by aspenberg showed that while corticosteroid injection has short term benefits , in the long term it is more likely to cause harm . however , there are others who believe that corticosteroids are a poor treatment choice for lateral epicondylitis . in order to address treatment concerns for patients with lateral epicondylitis ; we did a randomized double - blind clinical trial to test the effectiveness of steroid injection versus placebo and immobilization versus no immobilization in treating patients with lateral epicondylitis . this randomized double - blind clinical trial was conducted in the kashani university hospital in isfahan , iran during the first 6 months of the 2013 . patients with confirmed lateral epicondylitis , who had not received any treatment prior to enrolment , were entered into the study if they had none of the exclusion criteria and gave informed consent for participation [ figure 1 ] . patients were seleted from a pool of individuals who were screened for upper extremity complains , those who were suspected of suffering from lateral epicondylitis were examined by an orthopedic surgeon and if a diagnosis of lateral epicondylitis was confirmed and none of the exclusion criteria were present , then they were asked to participate in an orientation session where the trial was explained to them and information booklets were distributed between them . those patients who agreed to participate in the trial were asked to give written consents . the inclusion criteria included a history compatible with lateral epicondylitis and positive examination in palpation of the elbow over the lateral epicondyle , with resisted wrist extension , resisted middle finger extension and/or the mills test , and patients with symptoms lasting more than 6 weeks , no history of acute trauma , fracture , and/or surgery within 12 months , patients who had not received corticosteroid injection , physiotherapy , splint or casting during the past 6 months , no bilateral involvement and no history of cervical disk herniation , radiculopathy or abnormal electrophysiologic study . random numbers table was used to allocate patients between groups 1 , 2 , 3 and 4 . the trial pharmacist also prepared a series of similar vials containing either 40 mg of depomedrol ( aburaihan comp . , iran ) ( 1 cc ) or 1 cc normal saline and coded them either 1 , 2 , 3 or 4 , the group assignments were not decoded until the end of the trial when the final analysis was due to take place . patients took their envelopes to the trial pharmacist who gave them a coded vial which they took to the orthopedic surgeon who made the injection . due to the color difference of depomedrol and normal saline , both vials and syringes were covered by stickers in order to conceal the injection solution . maximum point of tenderness was identified and injection was performed in fanlike fashion in that area . after the injection the trial clerk took the patients to a technician who gave patients in groups 1 and 3 long arm splints . the trial subjects were then evaluated using oxford elbow scale ( oes ) and visual analog scale ( vas ) ( oes is the gold standard for clinical evaluation of elbow complaints ) by the trial clerk . the patients were evaluated at the baseline and before administration of treatment and they were also asked to come to the trial office at 2 weeks , 4 weeks and 24 weeks for follow - up evaluation which was also conducted by administration of oes and vas by the trial clerk . oes is a patient reported questionnaire consisting of 12 questions that has been shown to have high specificity and sensitivity in assessing the outcomes of interventions on the elbow of the patients . these questions assess three domains pertaining to the elbow : function , pain and social - psychological with four questions for each domain . the questionnaire was translated into persian by a professional translator and was then translated back into english in order to ascertain accuracy . the translated questionnaire was then validated using a group of 20 random individuals and its reliability was tested by retest of the same individuals ( which showed 95% reliability ) . after all of the enrolled patients finished the 24 weeks follow - up , the data was entered into an ibm spss database ( ver . 18 ) and analyzed . the groups were subsequently decoded so that the interventions used in each group become known . due to the number of groups and multiplicity of comparisons made , we used bonferroni correction in order to adjust the p value accordingly . this study was approved by the ethics committee of the isfahan university of medical sciences ( protocol record 387278 ) and funds were provided by the university research council . overall in the 6 months period of recruitment , 91 patients which met the inclusion and exclusion criteria were identified and from this pool of patients , 79 accepted to participate in the study . at the end of the 24 weeks follow - up , 78 patients had finished the study with one patient lost to follow - up . after decoding the groups it became known that group 1 had received 40 mg depomedrol along with long arm splint , group 2 had received 4 mg depomedrol alone , group 3 had received normal saline with splint and finally group 4 had received normal saline injection only . there were 21 , 19 , 19 and 20 test subjects in the first , second , third and fourth group , respectively . one of the patients in the second group was lost to the follow - up after the initial treatment and was subsequently excluded from the final analysis . chi - square test failed to show any significant difference between the groups with regards to gender distribution ( p = 0.866 ) . the average age of the participants was 47.39 years ( standard deviation [ sd ] : 6.53 , range : 32 - 65 ) ; repeated measure analysis of variance ( anova ) test did not show any significant difference in the age composition of the subjects of the study groups ( p = 0.622 ) . distribution of variables between the four groups the average vas score of the participants prior to the administration of the treatment was 5.93 ( out of ten , sd : 0.93 ) and no significant intergroup difference was noticed ( p = 0.577 ) . the average oxford elbow score of the patients prior to receiving treatment was 21.21 ( sd : 2.80 ) again repeated measure anova test failed to show any intergroup difference ( p = 0.658 ) [ table 1 ] . before and after tests showed that all groups reported better outcomes at different follow - up intervals and at the end of the trial . in order to understand which treatment was more effective we compared the mean differences of the groups outcomes at different stages of the trial . we measured the difference between the mean vas score at different stages of the trial [ figure 2 ] and then compared these means differences between the four groups . the analysis showed that at the 2 week follow - up , both of the corticosteroid injection groups were similar ( p = 1 ) but the patients in these two groups had a significantly larger reduction in the vas score compared with the two groups which received normal saline injection ( p < 0.001 ) . in the groups which received normal saline injection the patients in group 4 who received long arm splints in addition to normal saline injection fared better ( mean difference : 0.79 , p = 0.005 ) . at 4 weeks follow - up , again the corticosteroid injection groups were similar but significantly better than the normal saline injection groups ( p < 0.001 ) but by this time the two normal saline injection groups had become similar ( p = 0.768 ) . by the 24 week , a reversal of results was observed as the groups who received saline injection reported better results ; at 24 weeks , the two corticosteroid injection groups were similar ( p = 0.372 ) . the group who received depomedrol alone at 24 weeks reported worse scores than the normal saline only injection group ( p = 0.044 ) but they did not report significantly different outcomes compared to the normal saline and splint group ( p = 0.847 ) . the group that received both corticosteroid injection and long arm splint , however , were significantly worse than both of the groups that received normal saline injection ( p < 0.01 ) . mean visual analog scale of the four groups during the 24 weeks of study we measured the difference between the mean oes score at the beginning and the end of the trial [ figure 3 ] and then compared these means differences between the four groups . the depomedrol only group reported similar scores to that of the depomedrol and splint group ( p = 1 ) but they reported lower scores compared to the both normal saline injection groups ( p < 0.05 ) . the depomedrol and splint group reported similar scores to the normal saline and splint group ( p = 0.157 ) but reported worse scores compared to the normal saline only group ( p = 0.025 ) . the two groups that received normal saline injection reported similar scores ( p = 1 ) . mean oxford elbow scale of the four groups at beginning and at week 24 none of the patients were retreated or sought further treatment in the 6 months period of follow - up and as the results showed regardless of the intervention used the patients all reported a degree of improvement compared to their baseline status . lateral epicondylitis is believed to have three distinctive phases : the initial phase , the subacute phase and the chronic phase . in the first two stages management options include icing , rest , physical therapy and bracing with 95% of patients healing spontaneously with minimal intervention . there is , however , no consent on management of chronic epicondylitis . because of high rate of spontaneous healing , watchful waiting has been proposed as an initial approach with some even arguing that this may be the best approach overall to lateral epicondylitis . non - surgical treatment of lateral epicondylitis is a highly debated issue ; over the years many treatment modalities have been proposed but there has been no conclusive evidence to show that either of them works best for this condition . a systematic review by bisset et al . showed that while enough evidence does not exist ; there may be a short term benefit for physical joint manipulation and exercise but evidence for both of these was limited and lacked long term follow - up . ultrasound , ionization and acupuncture were also shown to be beneficial up to 3 months , but after a year of follow - up their benefit disappeared . their results also showed that laser therapy and extracorporeal shockwave therapy have no beneficial effect either in short term or long term . there are , however , more recent trials that have shown some effectiveness for radial shock wave therapy in cases where other modalities have failed . topical non - steroidal anti - inflammatory drugs may also be beneficial in reducing pain in patients . while over the years , corticosteroid injection was debated as one of the choices for treatment of lateral epicondylitis , in the recent years , the evidence has pointed that corticosteroids , at least in the long run , have limited benefit or are even harmful and cause recurrences . the evidence shows short term ( 1 month ) benefit for either corticosteroid injection or physiotherapy alone ( not both ) but this benefit fades after the early months . recent meta - analysis of the available evidence has confirmed the short term benefits of corticosteroid injection , but in the long term , the adverse effects have been shown to far outweigh the short term benefits of this treatment modality . again showed that corticosteroid injection in the short term causes pain alleviation but this effect reverses over long term . they also showed that there may be short term benefit for the patients who are treated with sodium hyaluronate injection , botulinum toxin injection or prolotherapy . compared steroid injection with injection of autologous blood for treatment of patients with lateral epicondylitis ; both treatment modalities were equally effective at 2 weeks but at 6 weeks it was shown that autologous blood injection has a significantly larger effect than steroid injection but more studies are needed for evaluation of this treatment modality . overall , more recent reviews of injection modalities have shown effectiveness for platelet rich plasma , botulinum toxin , hyaluronic acid and prolotherapy but the trials that reported clear benefits for these modalities were at high risk of bias with the exception of prolotherapy . more recent studies have also shown benefit associated with prolotherapy in treatment of lateral epicondylitis . in a more recent study by krogh et al . , it was shown that steroid is more effective in pain reduction at 1 month compared to either saline injection or platelet rich plasma injection , but in 3 months no clear advantage for either method was noticed . furthermore , it was shown that corticosteroid injection leads to reduced color doppler activity and tendon thickness compared to the other modalities which puts the patients at a higher risk for adverse effects . the high risk of recurrence with corticosteroid injection was again documented in a trial by mardani - kivi et al . where they compared corticosteroid injection with procaine injection . corticosteroid injection also has many documented adverse effects the most serious of which is tendon rupture . these adverse effects have been reported in most of the clinical trials that evaluated the effectiveness of corticosteroid injection . there seems to be adverse effects for all injection treatment modalities with the exception of sclerosant and platelet - rich plasma injections , which on the other hand have shown no clear beneficial effect either . our results have shown that when measuring the pain of the patients using the visual analog scale , they report a clear short term benefit when treated with corticosteroid injection ; namely injection of 40 mg depomedrol , but this benefit fades by the 24 week of follow - up . there is , also , no obvious pain relief advantage when splinting is added to the treatment modality . the advantage of corticosteroid injection is lost , however , when the more comprehensive oes is used ; oes combines questions about pain , with those inquiring about function and psychosocial aspects . our results show that when comparing oes scores at 24 weeks , patients who had received normal saline injection reported better scores compared to the group which received corticosteroid injection , which might be related to the rebound effect of the corticosteroid injection . our results are consistent with other similar studies in that they show a clear pain relief benefit for corticosteroid injection in the short term , we have shown that this pain relief benefit over placebo lasts through the 6 month follow - up , the advantage is , however , reduced over time and by the 24 week the difference between the groups becomes minimal . on the other hand , the superiority of saline injection at 6 months compared to corticosteroid injection when the oes scale is used shows that while patients who receive steroid injection may have greater pain relief ; it seems that in other aspects , the possible unwanted effects of corticosteroids ( such as reduced tendon thickness ) causes them to have a lower oes score . we , however , did not observe any clinically significant adverse effect in the patients . our results indicate that despite the clear pain reduction benefit associated with steroid injection in short term , this benefit in comparison with normal saline injection fades by the 24 week of follow - up . all authors have assisted in preparation of the first draft of the manuscript or revising it critically for important intellectual content . all authors have read and approved the content of the manuscript and confirmed the accuracy or integrity of any part of the work .

sequencing bacterial genomes has traditionally required large amounts of genomic dna ( ~1 g ) . there have been few studies to determine the effects of the input dna amount or library preparation method on the quality of sequencing data . several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input dna . in this study , we evaluated the nebnext ultra library preparation reagents for sequencing bacterial genomes . we have evaluated the utility of nebnext ultra for resequencing and de novo assembly of four bacterial genomes and compared its performance with the truseq library preparation kit . the nebnext ultra reagents enable high quality resequencing and de novo assembly of a variety of bacterial genomes when using 100 ng of input genomic dna . for the two most challenging genomes ( burkholderia spp . ) , which have the highest gc content and are the longest , we also show that the quality of both resequencing and de novo assembly is not decreased when only 10 ng of input genomic dna is used . background : lateral epicondylitis is a common problem affecting 1 - 3% of the population . there has been much debate about the best treatment modality for this condition . there is , however , no conclusive evidence in support of any of the proposed treatment modalities . in this trial , we have studied the effect of corticosteroid injection ( with or without splinting ) with normal saline injection ( with or without splinting).materials and methods : in this double - blind , randomized clinical trial , individuals were randomly assigned to either of four treatment groups and received either 40 mg depomedrol injection alone , 40 mg depomedrol injection with splinting , normal saline injection alone , or normal saline injection with splinting . they were evaluated using the visual analog scale ( vas ) at weeks 2 , 4 and 24 and with the oxford elbow scale ( oes ) at 24 weeks.results:a total of 79 patients were participated in the study . the corticosteroid injection groups had better pain relief as measured by vas at 2 and 4 weeks compared with the two saline injection groups . mean vas difference at week 0 versus week 2 was 4.5 0.9 and 2.8 0.6 in corticosteroid injection groups and saline injection groups respectively ( p < 0.01 ) but at 24 weeks , there was only moderate benefit reported for the group which received steroid injection and splinting ( p < 0.01 ) compared to the saline injection groups . the saline injection groups reported better improvement in oes scores ( 20.1 3.7 ) at the end of the trial compared corticosteroid injection groups ( 16.1 2.9 ) ( p < 0.05).conclusion : our results indicate that despite the clear pain reduction benefit associated with steroid injection in short term , this benefit in comparison with normal saline injection fades by the 24th week of follow - up .

multiple sclerosis is a chronic demyelinating disease of the central nervous system , affecting any part of the cns , but mostly white matter tracts in the cerebral hemispheres , optic nerves , brainstem , cerebellum , and spinal cord . the prevalence of the disorder ranges between 2 and 150/100,000 depending on the country or specific population , affecting approximately 1 million people worldwide . psychiatric symptoms in the context of ms were first noted by charcot , who described mania , hallucinations , and depression among other manifestations of the disease . lifetime prevalence of major depression ranges from 19 to 54% , depending on the population sample and diagnostic criteria used [ 68 ] . annual prevalence is estimated at 16% , and point prevalence of clinically significant depressive symptoms is as high as 50% . the rate of depressive disorders in a pediatric population with demyelinating disease has been estimated at 27% . evidently , prevalence is considerably higher than in the general population or among patients with general medical conditions other than ms . etiologic factors seem to be both biological and psychosocial , and risk factors have been reported to be female sex , age < 35 years , family history of major depression , and a high level of stress . disease - modifying agents , interferon beta in particular , have also been suspected to induce depressive symptomatology [ 14 , 15 ] . however , subsequent well - designed studies failed to prove a depressogenic effect , and pretreatment depressed mood was found to be the best predictor of subsequent depression [ 16 , 17 ] . the importance of this clinical issue can not be underestimated , given its high frequency , the possible adverse effects of depression on disease course , cognitive functioning , treatment adherence , deleterious consequences on quality of life , and increased risk of suicide [ 22 , 23 ] . unfortunately , mental comorbidity and depression in particular are frequently underdiagnosed and undertreated , with reported rates of missed diagnosis around 2330% [ 6 , 24 ] and rates of inadequate treatment around 20%36% of those reporting depression [ 6 , 25 ] . in a cross - sectional community study of patients with ms by cetin et al . , 59% of the patients who had significant symptoms of depression were not taking medication , possibly due to missed diagnosis , denial of symptoms , or false reporting , whereas the rest 41% received inadequate treatment . in the whole sample ( n = 542 ) , 28% of the patients were adequately treated for depression and therefore had achieved remission . in another study by mohr et al . , 65.6% of depressed patients did not receive medication , 4,7% received subthreshold treatment , 26,6% received threshold , and only 3.1% received overthreshold , indicating that in most cases , therapy is not in accordance with treatment guidelines for depression . given the fact that depression is a treatable condition , correct diagnosis and adequate treatment are issues of immense importance for the management of multiple sclerosis patients . the present paper is focused on current knowledge on diagnosis , assessment , and therapeutic interventions for depression in the context of multiple sclerosis . although the symptom of depressive mood is almost always experienced by patients suffering from disabling diseases , the syndrome of major depression , as defined by the classification systems of icd-10 and dsm - iv - tr , corresponds to a constellation of symptoms comprising depressive mood , anhedonia , fatigue , psychomotor retardation or restlessness , suicidal ideation or suicide attempt , feelings of guilt and worthlessness , and difficulty in concentrating , as well as vegetative symptoms , including altered sleep , appetite , and sexual arousal . a common challenge for correctly diagnosing depression in the context of multiple sclerosis is distinguishing whether a certain symptom emanates from a depressive disorder or can be attributed to the demyelinating disease . potential confounders are fatigue , insomnia , altered appetite , and impaired memory and concentration , and misjudgments will lead to false positives or false negatives . varied presentations of depression , for example , unexplained somatic complaints , anxiety , or hopelessness instead of sadness , could also complicate diagnosis , especially in older persons . patients with ms might exhibit pathological laughter or crying occurring with bilateral forebrain disease , which poorly correlates with underlying mood . on the other hand a way of disentangling the differential diagnosis in such cases is by paying attention to the cognitive and affective domains of the depressive symptomatology , for example , the depressive mood and depressive beliefs , which is also an approach adopted by assessment tools and rating scales for depression in the presence of a physical illness and multiple sclerosis in particular . the use of two screening questions has been proposed for detecting major depressive disorder in the context of ms and primary care setting , which correspond to depressive mood ( during the last two weeks , have you often been bothered by feeling down , depressed , or hopeless ? ) and anhedonia ( during the past two weeks , have you often been bothered by little interest or pleasure in doing things ? ) . at least one affirmative response on either question was used as a criterion of having mdd . this approach was shown to have a positive predictive value of 71.7% , with a rate of 27.3% of false positives . however , two thirds of false positives were shown to have subthreshold depression , which again should alert the clinician for the need of treatment . using the yale single question is even less time consuming , but this tool seems to lack sensitivity , as it could not identify 34.7% of patients who were depressed by beck depression inventory criteria . on the other hand , it appears to be quite specific , that is , depression can be ruled out when it does not exist . the beck depression inventory is an objective self - report assessment tool comprising 21 items , and one of the most commonly used for patients with multiple sclerosis . recommended cutoff point is set at 13 , though 30% of patients with depression will be missed in this way . concerns regarding the length of the instrument as well as the inclusion of items corresponding to neurovegetative symptoms , which might lead to overdiagnosis , have led to the implementation of shorter forms , such as the 7-item beck depression inventory - fast screen ( bdi - fs ) . validity of the instrument has been documented for the population of patients with multiple sclerosis . the hospital anxiety and depression scale is a self - assessed questionnaire consisting of 14 items , suitable for use in persons with medical conditions , if items corresponding to somatic symptoms are omitted , and recently validated for people with multiple sclerosis . the center for epidemiologic studies depression rating scale ( ces - d ) , comprising 20 items , has been used with a cutoff point of 16 ( > 15 ) , as likely to correspond to significant depression , recognizing 74.5% of depressed persons [ 9 , 45 ] . another useful scale is the chicago multiscale depression inventory that was developed to assess depression in ms and other chronic diseases . it is a 42-item self - reporting questionnaire consisting of three subscales , mood , physical malfunctioning , and self - criticism , the first of which is recommended for ms . the validity of the use of the hamilton rating scale for depression has been examined in one study . the authors conclude that the whole or part of the scale can be used , depending on study design . the depressive mood scale ( ehd , echelle d ' humeur dpressive ) is an 11-item french questionnaire specifically designed and validated for the assessment of depression in ms , focusing on mood changes rather than somatic symptoms . the zung self - rating depression scale ( zsrd ) is a 20-item tool that has been shown to have good construct validity for measuring depression in medically ill populations and has been used for patients with ms [ 53 , 54 ] , as well as the montgomery - asberg rating scale ( madrs ) , a widely known clinician - rated assessment tool for depression , consisting of 10 items . in pediatric populations , researchers most frequently use the child depression inventory for identifying depression in samples with ms [ 57 , 58 ] . the presence of depression can also be documented by applying the kiddie - sads interview . the utility of detecting depression partly depends on the efficacy and availability of treatments as well as willingness of patients to receive treatment . in a recent study , less than 30% of depressive individuals who were prompted to receive treatment did so , and possible explanations for this were distorted beliefs of depression being inevitable , practical problems , or the depression itself [ 60 , 61 ] . concluding , identifying depression , though not sufficient to ensure effective treatment , is a necessary first step towards proper management of depressed patients with multiple sclerosis . a comprehensive treatment plan for depression should include pharmacotherapy , psychotherapy , or cognitive behavioural therapy in specific or combination therapy . screening for suicidal intend is incremental due to the high cumulative lifetime risk for suicide . treating depression has been found to improve adherence , cognitive disturbances , fatigue , quality of life , and possibly disease course , by decreasing production of cytokine . there is a growing body of evidence regarding the neuroprotective effects of antidepressants such as fluoxetine and phenelzine . furthermore , escitalopram has been found to reduce stress - related relapses in a recent open - label , randomized , controlled study . treatment guidelines have been most recently reported by the canadian network for mood and anxiety disorders ( canmat ) , based on up - to - date literature . use of antidepressants is strongly recommended , as well as psychotherapies emphasizing coping strategies rather than insight . drugs with significant sedating or anticholinergic side effects , such as tricyclic antidepressants , should rather be avoided , due to issues with fatigue , orthostatic hypotension , balance , cognitive disturbances , and bladder problems . patients should also be monitored for manic or hypomanic symptoms , while on antidepressant medication , as the prevalence of bipolar disorder is quite elevated in ms , presumably two to three times higher than in the general population .should depression emerge during treatment with interferon beta , the latter needs not necessarily be discontinued , but the depression should be adequately treated instead . despite the high burden of depression in multiple sclerosis patients , few trials have been published regarding this particular population , and only two of them were double blind , randomized , placebo controlled , and meeting certain standards of quality , as already observed by koch et al . . the first one examined the efficacy of desipramine versus placebo and found a trend in favor of desipramine . the second demonstrated greater efficacy of paroxetine versus placebo , with 78.6% versus 42.1% of patients exhibiting response , respectively , but the difference did not reach significance , probably because of underpowering of the study and dosing and duration issues . another problem faced by both studies was missing data or patients who were lost at followup . in another study by mohr et al . , sertraline was found to be equally effective with cognitive behavioural therapy , and both of them were more efficacious than supportive - expressive therapy in a sample of 63 depressed patients with multiple sclerosis . open - label studies have demonstrated efficacy of sertraline , fluvoxamine , moclobemide , and fluoxetine . a few case reports deal with the usefulness of ect for severe or refractory depression in ms ; however , concerns have been raised that ect might mediate neurological deterioration by altering the number or size of the cns plaques and/or periplaque edema [ 79 , 80 ] . it has been postulated that the presence of contrast - enhanced lesions might predict deterioration , and therefore , a gadolinium - enhanced mri is suggested , but this observation is based on a report of only three patients , one of whom deteriorated after ect sessions . psychotherapy has long been considered an important treatment option for the management of depression in patients with ms , with approaches focusing on coping skills showing superiority over insight - oriented therapies . cognitive behaviour therapy ( cbt ) helps patients to correct distorted cognitive appraisal of the environment and core beliefs that lead to maladaptive behaviour , and change the connection between life events and learned reactions such as depression beliefs . regarding multiple sclerosis in particular , which imposes progressively increasing physical difficulties and challenges in everyday living , together with cognitive impairment , cbt can help maximize the development of the patients ' coping skills . in line with this observation , individual ( cbt ) has been found more beneficial than supportive expressive therapy ( set ) , administered either as usual or by telephone . telephone - administered psychotherapy or counseling is particularly appealing because of the frequent physical disability , which represents an obstacle to receiving therapy otherwise . for similar reasons , computerized forms of cbt are also available [ 86 , 87 ] . , there still may be some patients who would benefit more by insight - oriented , psychodynamic psychotherapy , which focuses on the management of psychological defenses and unconscious psychic activity [ 82 , 88 ] . the effectiveness of mindful - based intervention ( mbi ) which is based on the nonjudgmental awareness of everyday moments has also been recently demonstrated . individual preference and needs , cognitive performance , and availability of therapist should help the clinician choose among the above diverse psychotherapeutic approaches . the prevalence of depression in patients is remarkably high , yet it is still frequently underdiagnosed and undertreated . patients who are not treated are not expected to improve and are at risk for further deterioration . more and better designed studies for therapeutic interventions , particularly in the pediatric populations , are clearly needed . variability in clinical response to standard therapeutic dosage regimen was reported in the 1950s by many pioneers in the field . since then , the association between monogenic polymorphisms and variations of drugs ' metabolism , transport , or target had been identified and the vision of personalized drug therapy in health care envisioned [ 1 , 2 ] . pharmacogenomic - guided drug therapy for patient is based on the premise that a large portion of interindividual variability in drug response ( efficacy and/or toxicity ) is genetically determined . despite the widespread recognition of the scientific rationale and the clinical implementation of pharmacogenomic tests at several major academic medical institutions [ 37 ] , most clinicians and researchers engaged in the discipline would agree that the early vision of achieving personalized therapy in the form of therapeutic regimens tailored to an individual 's genetic profile remains some years away . broadly speaking , the development and implementation pathways for pharmacogenomic tests consist of several stages ( figure 1 ) : first , discovery of pharmacogenomic biomarkers and validation in well - controlled studies with independent populations ; second , replication of drug - gene(s ) association and demonstration of utility in at - risk patients ; third , development and regulatory approval of companion - diagnostic test ; fourth , assessing the clinical impact and cost - effectiveness of the pharmacogenomic biomarkers ; fifth , involvement of all stakeholders in clinical implementation . lessons learned in making pharmacogenomic - guided therapy useful to clinicians have identified multiple scientific challenges and implementation barriers existing within these stages , each of which is fueled by multitude of stakeholders with varied goals and interests . this paper will provide a perspective on these existing challenges and barriers in the complex process of implementing pharmacogenomics in clinical practice , as well as incorporating pharmacogenomics into the drug development process . many pharmacogenomic biomarkers have been identified over the last decade , but only few of them have been utilized to different extents in clinical setting ( table 1 ) . one of the major challenges for translating most discovered biomarkers to their clinical implementation as genomic tests has been the inconsistent replication result of genetic associations , whether alone or in combination . traditionally , the candidate gene approach incorporating a panel of genes that encode known drug targets , metabolizing enzymes , and membrane transporters is used in pharmacogenomic studies to test the hypothesis of an association between single nucleotide polymorphisms ( snps ) and a pharmacological or therapeutic endpoint . a good example of inconsistent replication result of genetic associations is the atypical antipsychotic clozapine with its complex pharmacological effects via the dopaminergic , serotonergic , adrenergic , and histaminergic receptors within the central nervous system . over the years , conflicting study results exist in the literature for association between clozapine response with either snps of each known pharmacological receptor subtype [ 1013 ] , combinations of polymorphisms , and metabolizing enzymes and transporters . it is also of note that the original association regarding combination of polymorphisms was not replicated in a subsequent study . the recent identification of yet another new candidate gene for clozapine treatment response illustrates the limitation of candidate gene approach in that there is always the possibility of involvement of other yet - to - be - identified genes , including those that have not been known to be linked to the pharmacology of the drug , that could account for additional variability in patient 's therapeutic response . when evaluated or used alone , most of these markers are likely of insufficient sensitivity and specificity to provide clinically useful prediction , especially of efficacy . the recognition of multiple gene variants , rather than snps , each accounting for part of the disposition and response phenotypes , has led to the increased use of whole genome approach for discovery of new biological pathways and identification of associations between pharmacogenomic biomarkers and response phenotypes . genome - wide association study ( gwas ) approach screens large number of snps ( up to 2.3 million per array ) across the whole genome in order to determine the most significant snps associated with response phenotypes . in contrast to the hypothesis - driven candidate gene approach , there is no a priori knowledge of specific gene for the discovery - driven gwas approach . rather , the large numbers of snp analyses test multiple hypotheses and necessitate large sample size , sophisticated computing and platforms ( e.g. , affymetrix genechips ) , and high cost . in addition , the level of significance associated with each test needs to be corrected for multiple hypothesis testings . refinement of the gwas approach takes a two - step design , using high - density array to discover the snp associations in a population cohort followed by replicating the initial findings above the genome - wide significance with additional patient sets in a more hypothesis - driven study of sufficient sample size . while this approach has been successfully applied in the pharmacogenomics of clopidogrel , flucloxacillin , simvastatin , and warfarin [ 1722 ] , the implications of the results are less clear for other drugs such as the psychotropics [ 2330 ] . a middle - of - the - road approach would be to limit the number of snps that warrant analysis . based on the phenomenon of linkage disequilibrium among snps , whereby two or more snps are inherited together in haplotype blocks more frequently than would be expected based on chance alone , a single representative snp within a haplotype block could serve as a tag snp ( tsnp ) for the haplotype . by genotyping a smaller number of carefully chosen tsnps to identify haplotype blocks of dna sequences that are inherited together the hapmap database created by the international hapmap project ( http://www.hapmap.org/ ) is freely available for selection of these tsnps . based on the hapmap database , many gwass of drug responses have been completed [ 18 , 19 , 32 , 33 ] . it is hoped that some of the scientific challenges for study replication related to snp genotyping may be alleviated through this approach . regardless of the choice of approach to identify the genotype - phenotype association , population variations in prevalence and relative importance of different allele variants , for example , cyp2d6 , hla - b , ugt1a1 , and slc6a4 , remind investigators of the importance of ethnicity and population stratification [ 35 , 36 ] , which could magnify the sample size requirement for statistical power in most pharmacogenomic studies . for example , although the algorithms based on the work of gage et al . and the international warfarin pharmacogenetics consortium ( iwpc ) [ 38 , 39 ] are clinically useful , they do not include detection of the cyp2c98 , an allele commonly occurring in african americans . the lower success with algorithm - based dose prediction in african americans is likely related to exclusion of this allele in most dosing algorithms . another example is hla - b1502 being a strong predictor of carbamazepine - induced severe cutaneous drug reactions in han chinese and most southeast asians but not in caucasians , who do not carry the allele variant [ 4143 ] . if not accounted for , these ethnicity - or population - related variables will confound the results of most pharmacogenomic association studies and could complicate the result interpretation . in addition , there is no universal agreement among different test platforms as to which allele variant should be tested routinely for some genetic polymorphisms , for example , cyp2d6 and ugt1a1 . in addition to the aforementioned ethnicity - related considerations , the drug disposition and response phenotypes can be affected by patient - specific variables . phenocopying with a change in metabolic phenotype secondary to concurrent enzyme inhibitor [ 44 , 45 ] could create genotype - phenotype discordance and affect the ability to predict possible drug response based on genotype - guided dosing and achievable drug concentration . inflammatory responses elicited by extrahepatic tumors have been shown to release cytokines such as interleukin-6 ( il-6 ) and resulted in transcriptional downregulation of the human cyp3a4 gene . therefore , lower docetaxel clearance reported in cancer patients could be related to tumor - associated inflammation and subsequent transcriptional repression of cyp3a4 , potentially leading to unanticipated toxicity despite normal enzymatic activity in the patient . il-6-mediated downregulation of cytochrome p-450 enzyme activities also likely contributed to a recent report of significant increase in clozapine concentration in a patient with infection and inflammation . an additional challenge for applying pharmacogenomic biomarkers in targeted cancer therapeutics is sampling of tumor tissue that carries the somatic mutations ( e.g. , testing for the epidermal growth factor receptor 1 ( her1 ) mutation in patients treated with gefitinib for nonsmall cell lung cancer and testing for overexpression of the human epidermal growth factor receptor 2 ( her2 ) protein in patients receiving trastuzumab for breast cancer ) . the presence of tumor cell heterogeneity might result in intra- and interindividual variabilities in tumor tissue content and , hence , measurable level of the biomarker . in spite of this limitation , furthermore , there is an increasing appreciation that genetic heterogeneity alone can not explain interindividual variations in drug responses . yet currently , much less is known about the influence of environmental variables and gene - environment interactions on drug disposition and response phenotypes such as mutations and polymorphisms [ 4951 ] . environmental factors , through their participation in epigenetic mechanisms , could result in many different phenotypes within a population . in the not too distant future , pharmacoepigenetic investigations focusing on studying the interaction among drugs , environment , and genes could provide additional insight of drug response variations beyond the level of genetic polymorphisms . after demonstration of a genetic association with response phenotype , there is the need of validating the biomarker , regardless of whether it is to be developed as a companion diagnostic test . for the purpose of personalized therapy , a companion diagnostic for a drug can be defined as a biomarker that is critical to the safe and effective use of the drug . the acce ( analytical validity , clinical validity , clinical utility and associated ethical , legal , and social implications ( elsi ) ) model project sponsored by the office of public health genomics , centers for disease control and prevention ( cdc ) , has been recently advocated by some investigators to be the basis for evaluation of pharmacogenomic biomarker tests . analytical validity determines how well a diagnostic test measures what it is intended to measure , regardless of whether it is an expression pattern , a mutation , or a protein . clinical validity measures the ability of the test to differentiate between responders and nonresponders , or to identify patients who are at risk for adverse drug reactions . the clinical utility measures the ability of the test result to predict outcome in a clinical environment and the additional value over nontesting , that is , standard empirical treatment . in 2004 , the cdc launched the evaluation of genomic applications in practice and prevention ( egapp ) initiative , which aims to establish an evidence - based process , including assessments of analytical validity , clinical validity , and clinical utility , for evaluating genetic tests and genomic technology that are being translated from research to clinical practice . for the pharmacogenomics discipline , one often - cited publication was the 2007 egapp working group evidence - based review of the literature on the use of cyp genotyping for clinical management of depressed patients with the selective serotonin reuptake inhibitors ( ssris ) . based on strong evidence of analytical validity , possible demonstration of clinical validity , and lack of study data to support evaluation of potential clinical utility , the working group does not recommend the application of cyp2d6 genotyping for ssri pharmacotherapy . since approval of most cyp genotyping tests by the food and drug administration ( fda ) is dependent on their technical performance in detecting cyp450 gene variants , the weak evidence of association between genotype and phenotypes ( different metabolic phenotypes , responders versus nonresponders ) is also not unexpected , since most ssris rely on multiple but not necessarily polymorphic enzymes for metabolism and have a flat dose - response relationship with wide therapeutic index . the clinical validity of the cyp genotyping tests to differentiate response phenotypes is further limited by the cyp genotype - metabolic phenotype discordance that can occur as a result of drug - drug interactions [ 44 , 45 ] or environmental influences . given these limitations as well as the lack of cost - effectiveness data , it is not surprising that the ssris are not good candidates for genotype - based pharmacogenomic therapy and , hence , the recommendation of the egapp working group . other pharmacogenomic biomarkers could be better candidates for testing association between specific genotype and clinical phenotype [ 5563 ] , as indicated by published guidelines . pharmacogenetic dosing algorithms [ 37 , 39 ] based on the patient 's cyp2c9 and vkorc1 genotypes and other nongenetic factors ( e.g. , age , body size , and concurrent interacting drug ) have been used to determine warfarin dosage regimens . as shown for clopidogrel , simvastatin , and warfarin , replication of the association in multiple cohorts or inclusion of replication data would provide further evidence of clinical validity [ 17 , 18 , 64 , 65 ] . establishing the clinical utility of pharmacogenomic biomarkers has been advocated to ensure that their use is appropriate , cost - effective , and ultimately improves clinical outcome in patients . yet within the clinical and scientific communities , there are constant debates with little agreement regarding the required levels of evidence for proof of clinical utility of diagnostic tests that are scientifically appropriate but at the same time realistically achievable [ 6671 ] . the gold standard for demonstration of clinical utility of a drug is the use of randomized controlled trials ( rcts ) . given the current evidence - based driven clinical environment , many investigators advocate that hypothesis - driven , prospective , double - blind rcts would provide the ideal approach to validate the clinical utility of pharmacogenomic biomarkers . however , within the context of personalized medicine , the biomarker as a companion diagnostic test is intended for use with a drug to produce the optimal efficacy and safety . this makes it difficult to distinguish the clinical utility of the test that is different from that of the drug or the drug - test combination . in addition , the traditional assessment of evidence of drug efficacy and safety with the use of rcts may not necessarily portray the benefit of pharmacogenomic biomarkers . complex disease etiologies , heterogeneous patient population , placebo effects , and drug response variabilities per se all contribute to statistical power issues that necessitate large patient cohort for rct . all too often , the end result is achievement of small average benefit in the entire heterogeneous patient cohort , despite the trial being costly in terms of time and sample size . in contrast to evidence - based practice , the emphasis and value of pharmacogenomics are more geared towards incremental advantages in efficacy and safety for the outliers ( the poor metabolizers , the ultra - rapid metabolizers , the nonresponders , or those susceptible to develop adverse drug reactions ) over traditional therapy or standard dosing regimen . for example , the iwpc showed that a pharmacogenetic dosing algorithm was most predictive of therapeutic anticoagulation in 46% of the patients cohort who required < 25 mg / week or > 49 mg / week . therefore , a balance between the scientific demands of rcts and the practical value of genotyping for patient care seems appropriate . given the low prevalence of genetic variants associated with drug response and the desire to generate more robust evidence , many investigators and sponsors have advocated the use of prospective enrichment design clinical trials to include patients who are more likely to respond or at least be stratified according to disease subtypes and/or exclude patients who are highly susceptible to adverse drug reactions . however , even with the assumption of ( and sometimes proven ) association between genetic variabilities and drug response , both advantages and disadvantages exist for this study design . a recent simulation study of trial designs suggested that conducting more trials with smaller sample sizes and lessened evidence - based criteria might contribute substantially to cancer survival , and assessment relying solely on the current traditional , risk - averse trial design might slow long - term progress . in this regard , it is of note that the fda recently approved crizotinib and vemurafenib with their respective pharmacogenomic biomarker tests solely on data from two single - arm studies . examples would be prescribing of abacavir in patients tested positive for hla - b5701 and 6-mercaptopurine or azathioprine in homozygous carriers of tmpt mutations . likewise , conducting a pharmacogenomic add - on as part of a head - to - head efficacy comparison of two antipsychotics in patients who are carriers of the del allele of the 141c ins / del polymorphism in the dopamine d2 receptor gene would be difficult . the del allele is associated with poor antipsychotic response ; yet , all currently marketed antipsychotics are d2 blocker , albeit with different extent of blockade . not surprisingly , pharmaceutical companies have very little financial incentive to conduct time- and cost - intensive rcts , especially for out - of - patent marketed drugs . to move the discipline forward to eventual implementation , we have to rethink the types of study design and/or the quality of study data for evidence of clinical validity and utility . the concept of conducting practical clinical trials in real - world setting had been previously proposed for regulatory decision - making [ 77 , 78 ] . the recent study by anderson et al . provided evidence of comparative effectiveness between pharmacogenetic - guided warfarin therapy in 504 patients versus standard care in 1,866 patients and a strong validation to the clinical benefit associated with the use of pharmacogenomic biomarkers in a real world setting . at the grassroot level , the concept of practical clinical trial can even be modified and adopted on a much smaller scale in clinics or physician offices . as an example , elimination of tolbutamide is known to be 50% and 84% slower in carriers of cyp2c92 and cyp2c93 variants , respectively , than in homozygous carriers of cyp2c91 . yet , to - date , there is no prospective rct to evaluate the appropriateness of 50% to 90% dose reductions for patients who are carriers of the two allelic variants . in contrast , evaluating tolbutamide efficacy can be easily done after implementation of these dosage reductions . therefore , such effort in clinical practice , instead of expensive and time - consuming rct , could constitute the first step of obtaining evidence of clinical utility of cyp2c9 genotyping in optimizing tolbutamide therapy . for patient care , a good example for the need of balance between evidence - based medicine and personalized medicine is clopidogrel . despite the extensive evidence of clopidogrel efficacy linked to cyp2c19 genetic polymorphism [ 81 , 82 ] , debates continue over the routine use of cyp2c19 genotyping to guide clopidogrel therapy [ 8385 ] . this prevents more widespread use of the biomarker in individualized therapy , despite the significantly higher rates of stent thrombosis and the associated mortality rates in carriers of the reduced - function cyp2c192 allele . based on lack of outcomes data , the joint clinical alert issued in 2010 by the american college of cardiology and the american heart association did not recommend routine genotyping and suggested the need of large , prospective , controlled trials . one such trial is the pharmacogenomics of antiplatelet intervention-2 ( papi-2 ) trial that evaluates the effect of genotype - guided antiplatelet therapy versus standard care on cardiovascular events among 7,200 patients undergoing percutaneous coronary intervention ( pci ) ( clinicaltrials.gov nct01452152 ) . the questions then become are we in the meantime sacrificing patient care on the insistence of waiting for proof of value via the evidence - based approach ? if no study results are available in the near future , should we focus on steps that can facilitate the genotyping implementation in clinical setting and examine the cost - effectiveness of genotypes - guided antiplatelet therapy with a variety of different approaches ? for many healthcare facilities and systems , it is also critical to assess whether a test offers a good return on investment . therefore , in addition to clinical validity and clinical utility , another potential barrier to test implementation is demonstration of cost - effectiveness of the companion diagnostic test . ideally , the pharmacogenomic biomarker will result in cost - effective improved clinical care in patients who will benefit from individualized therapy with the drug and avoidance of cost - ineffective treatment for patients who likely will not benefit from the drug , either as a result of lack of response or increased adverse drug reactions [ 86 , 87 ] . traditional cost - effectiveness analysis compares the relative costs and outcomes of two different approaches , typically visualized on a cost - effectiveness plane divided into four quadrants . as mentioned in the last paragraph , avoidance of cost - ineffective treatment the antipsychotic drugs offer an alternative approach to cost - effectiveness evaluation for pharmacogenomics biomarkers . with an annual cost that is at least ten times higher , the atypical antipsychotic agents are more expensive yet no more efficacious and , hence , likely to be less cost - effective , than the typical antipsychotic agents [ 89 , 90 ] . rather than focusing on using biomarkers to predict efficacy of the more expensive atypical antipsychotic agents [ 1015 ] , genotyping for the glycine9 allele of the ser9gly polymorphism in the dopamine 3 receptor gene [ 91 , 92 ] might be used to identify patients susceptible to tardive dyskinesia , a highly prevalent adverse drug reaction associated with the use of the less expensive typical antipsychotic agents . the genetic testing might enable appropriate dose reduction for the typical antipsychotic agents and lessen the incidence of adverse drug reaction . additional approaches of demonstrating cost - effectiveness of pharmacogenomic - based therapy can range from clinical trial comparing per - patient cost for specific clinical outcome between genotype - based regimen and standard regimen to decision model - based study using simulated patient cohort [ 9496 ] . alternative approach exists even within the context of cost - effectiveness comparison between genotype - based regimen and standard regimen with no genetic testing . with generic availability of clopidogrel , a cost - effectiveness study of the value of pharmacogenomic biomarker should compare clopidogrel use in cyp2c19 ems and ums versus the use of prasugrel or ticagrelor for pms . regardless of the specific approach , it should be understood that the economic impact and cost - effectiveness of screening could be affected by different variables . two separate studies utilized modeling techniques with simulated patient cohorts to evaluate the potential clinical and economic outcomes for pharmacogenomic - guided warfarin dosing . while the relatively high cost of cyp2c9 and vkorc1 bundled test ( $ 326 to $ 570 ) resulted in only modest improvements ( quality - adjusted life years , survival rates , and total adverse rates ) , the investigators also suggested that improvements in the cost - effectiveness can be achieved in several ways , specifically further cost reduction of the genotyping test and utilizing genotype - guided warfarin dosing algorithm in outliers ( patients with out - of - range inrs and/or those who are at high risk for hemorrhage [ 97 , 98 ] ) . other variables such as different population prevalence of a specific variant and cost of alternative treatment approaches would also impact the economic impact analysis . in summary , clinical utility and cost - effectiveness can not be the only measures in determining the relative value of pharmacogenomics for drug therapy optimization in individual patients . rather , they should be used to supplement the best practice strategies currently in place to achieve optimal drug therapy . over the last decade , the fda has progressively acknowledged the importance of biomarkers and provided new recommendations on pharmacogenomic diagnostic tests and data submission . these efforts included the publication of fda guidance for pharmacogenomic data submission , guidance on pharmacogenetic tests and genetic tests for heritable markers , and draft guidance for in vitro diagnostic multivariate index assays ( ivdmias ) , the introduction of the voluntary data submission program , and formation of an interdisciplinary pharmacogenomic review group ( iprg ) to evaluate the voluntary submissions , as well as the approval and classification of different biomarkers . obviously , any biomarker with fda approval will generate more confidence for clinicians , healthcare facility administrators , and payers , and could enhance test implementation and utilization in the clinical settings . additional regulatory efforts also provide an impetus of pharmacogenomic data submission for drug approval and additional research to address the debate over the utility of the information incorporated in the revised labels , for example , for clopidogrel [ 8385 ] . within the united states , there are separate regulatory oversights for a pharmacogenomic biomarker developed as an in - house test by a clinical laboratory versus that for an in vitro diagnostic device developed by a medical device manufacturer . quality standards for clinical laboratory tests are governed by the clinical laboratory improvement amendments ( clia ) . in addition , the laboratories are accredited either by the college of american pathologists , the joint commission on accreditation of healthcare organizations , or health department of each individual state , that take into consideration of clia compliance and laboratory standard practices that are in line with good laboratory practice ( glp ) regulations enforced by the fda . although there is internal validation within the laboratory , there is no external regulatory review process for the test itself . on the other hand , although currently there is no formal regulatory process for submission of companion diagnostic tests , the fda previously ruled that evaluation and approval of the amplichip cyp450 test as an in vitro diagnostic device was required . in addition , the regulatory agency had fast track approved trastuzumab with the companion diagnostic hercep test in 2001 for detecting overexpression of her2 protein in breast cancer tissue by immunohistochemistry and more recently for tests that utilize fluorescence in situ hybridization to amplify the her2 gene . further examples of fda assuming a greater role were the respective companion diagnostic tests approved for crizotinib and vemurafenib . with the formation of a personalized medicine group within the office of in vitro diagnostic device , center for device evaluation and radiological health , it is likely that more fda - approved tests would be available in the future . although no similar frameworks for premarketing regulatory review and approval of pharmacogenomic biomarkers exist in the european union and the united kingdom , there are regulations applicable for postmarketing approval . gefitinib was approved by the european medicines agency ( ema ) in june 2009 , followed by subsequent approval of a companion diagnostic test for her1 mutations . there are several challenges and practical aspects related to clinical decision support infrastructure and training of healthcare professionals ( table 2 ) that need to be addressed before pharmacogenomic biomarkers can be successfully utilized in any healthcare setting . these are further discussed in the following sections . even with a decrease in genotyping cost over time , a relatively low demand for specific biomarker test at institutional clinical laboratories may not justify the cost of equipment and technical upkeep associated with in - house testing . this not only precludes the ideal point - of - care consultation at the bedside or within the clinic , but also results in long turnaround time for obtaining test results from external clinical laboratories or research institutions . the impact of the time delay would depend on the urgency of the test , for example , her2 expression or cyp2c19 genotyping prior to scheduled pci versus on - the - spot warfarin dosing adjustment or in the setting of emergency pci . a recent commentary of pharmacogenomics in primary care reported acceptable turnaround time of 24 hours for a feasibility study of warfarin pharmacogenetic testing in a family practice clinic . in addition , a point - of - care cyp2c19 genotyping device with a turnaround time of about an hour has been developed and recently used to explore the feasibility of incorporating cyp2c192 testing into clinical protocol for antiplatelet dosing . in addition to technology advances , the concept and adoption of preemptive ( preprescription ) genotyping [ 5 , 103105 ] with result stored in electronic medical record for subsequent use would also help minimize the inconvenience of time delay in test reporting . not unexpectedly , patients expect healthcare professionals to be able to explain the pharmacogenomic diagnostic test results and answer their questions regarding treatment access and choices . while interpretation of genotype result for deciding the appropriateness of a specific drug for a patient is usually not difficult , for example , the presence of the hla - b5701 variant for excluding abacavir therapy in patients with hiv-1 infection , the contrary would be true when the genotype result is used for dosing adjustment . the challenges for genotype - based doing guidelines are related to the multitude of genetic and nongenetic variables that can affect drug disposition and response , the significant interindividual variabilities in activities of most of the metabolizing enzymes , and the possibility of phenocopying with metabolic phenotype change in the presence of drug - drug interaction [ 44 , 45 ] . this difference in interpretation complexity related to the intended use of the test is likely one of the reasons for the fda to previously separate pharmacogenomic biomarkers into three categories . despite these challenges , warfarin dosing recommendations based on cyp2c9 and vkorc1 genotypes have been incorporated by the fda into the updated product label in 2010 . the dose table provided in the product label was reported to provide better dose prediction than empiric dosing [ 99 , 107 ] . however , the inclusion of most of the pharmacogenomics biomarkers as informational pharmacogenetic tests by the fda on the revised labels of many drugs , without clear guidance on dosing recommendation and/or therapeutic alternatives , usually results in a knowledge vacuum for the clinicians . all stakeholders would agree that lack of sufficient pharmacogenomics education for health professionals remains a major barrier for practical implementation of pharmacogenomics within the healthcare system . the need of adequate training was echoed in a recent usa survey of more than 10,000 physicians . although 98% of all respondents agreed that the genetic profile of a patient could influence drug therapy decision , only 29% had received some pharmacogenomics education during their medical training , and only 10% felt they were adequately trained to apply the knowledge in clinical practice . although the international society for pharmacogenomics recommended incorporating pharmacogenomics education in medical , pharmacy , and health science curricula , pharmacogenomics courses or materials have only been included to a variable extent at most pharmacy schools [ 110 , 111 ] . the gap in knowledge can currently be addressed through clinical guidelines available from professional organizations ( clinical pharmacogenetics implementation consortium , the international aids society - usa panel , the european science foundation , the british association of dermatology , and the pharmacogenomics working group of the royal dutch association for the advancement of pharmacy ) [ 5563 , 112 ] , availability of simple dosing algorithm such as that for warfarin , and further effort to include specific dosing recommendation in product label [ 99 , 107 ] . the most logical setting for initial implementation of pharmacogenomics would be healthcare facilities affiliated with academic institutions . the concept of pharmacogenomics - guided drug therapy is similar to that of clinical pharmacokinetics consultation service ( cpcs ) or therapeutic drug monitoring ( tdm ) program . in this regard , the familiarity of the cpcs or tdm program should be emphasized to clinicians who view the adoption of pharmacogenomics with some skepticism . likewise , hospitals with established cpcs or tdm program might find the task of introducing pharmacogenetic testing less formidable simply by expanding or modifying their existing clinical services . the availability of consultation service , in any format , should be complemented by educational training of clinicians to achieve specific competences . crews et al . reported significant increase in ordering of the cyp2d6 genotyping test one year after its availability via the cpcs . in a similar manner , once more clinicians are educated about the utility of pharmacogenomic approach to drug therapy , especially how to use the information , they would over time integrate pharmacogenomic findings and technologies into their practice . the importance of healthcare informatics for implementation of pharmacogenomics in clinical practice could not be overemphasized . at the level of patient care , integration of genotyping order template and/or genotype result into a robust system of electronic medical record ( emr ) with pop - up action alert and order templates for actionable pharmacogenomic tests to be used by physicians will be necessary [ 113 , 114 ] . at the level of research , the health information technology would enable organizational management of all research data and accessibility by the emr [ 115118 ] . both the patient care- and research - level informatics should incorporate updated information when available and be linked to other health informatics such as billing , clinical laboratory , and clinical trials within the healthcare facility . although adoption of emr is not universal , health information technology is a critical area for investment by healthcare system administrators , perhaps through collaborative efforts with the technology industry and the government . successful examples incorporating a coordinated team approach ( physicians , pharmacists , information technology and laboratory personnels ) with appropriate infrastructure support ( informatics ) to facilitate clinical implementation of pharmacogenomics have been reported at several institutions [ 35 ] . to fully integrate the multifacet process of the pharmacogenomics service , other organizational aspects of clinical decision support should include fostering effective communication and collaboration between laboratory staff and clinicians , creating flexible workflow with minimal disruption to the daily activities of the practitioners , delineating policies and reward systems that allow equitable schedule to minimize the additional time burdens perceived by some healthcare providers , and standardizing procedures to incorporate up - to - date pharmacogenomics - related information into formulary review and decision by the pharmacy and therapeutics committee . all these steps would facilitate implementation with minimal effect on work efficiency and cost for the healthcare system . successful implementation of pharmacogenomic biomarkers in clinical practice not only involves multidisciplinary coordination among physicians , pharmacists , clinical laboratories , health information specialists , and healthcare system administrators , but also requires collaborative efforts and willingness from the payer , a significant stakeholder in this endeavor . with the current healthcare landscape and the high cost of providing healthcare , the reimbursability of any particular test plays a significant role in deciding its implementation status in most healthcare facilities . while the cost of testing for several oncologic biomarkers and thiopurine s - methyltransferase in the united states is reimbursed in some hospitals , that is not the case for most pharmacogenomic biomarker tests . both federal and private payers are reluctant to reimburse the cost of the tests on the basis of either ( 1 ) lack of evidence of clinical utility ( which is usually associated with endorsement by professional organizations ) , ( 2 ) tests being not medically necessary ( because it has never been classified by the fda as required test ) , or ( 3 ) lack of cost - effectiveness analysis and/or comprehensive comparative effectiveness analysis . even with the product labeling information regarding the impact of cyp variants for warfarin , the centers for medicare and medicaid services recently denied coverage for genetic testing except when the test is provided for the purpose of clinical trials . this reluctance stance is consistent with the findings by cohen et al . who reported that most payers do not consider test accuracy in identifying subpopulations of interest , test cost , medication adherence , and off - label use as relevant factors in their consideration for reimbursement . in their survey of 12 payers , the most consistent determining factor is conclusive evidence linking the use of the diagnostic test with health outcome . even though most payers understand the implications of pharmacogenomics in healthcare and the potential return on investment , their reluctance to pay for diagnostic tests costing much less ( most costing $ 500 ) than what they actually pay for the more expensive drugs ( for which the diagnostic tests could be useful ) primarily reflects their expectation of demonstration of clinical utility and comparative effectiveness [ 120 , 121 ] . accordingly , inconsistent assessment of clinical utility and benefit could only result in confusion regarding the appropriate use and interpretation of biomarker - based pharmacogenomic diagnostic tests . hopefully , more realistic clinical practice guidelines from diverse groups of organizations and expert panels that take into consideration of the issues discussed earlier , would pave the way to greater extent of implementation . to that end , it is of note that regulatory guidance has been published to support the recommendation of the clinical practice guidelines . in addition , additional clarification from regulatory agencies regarding definition of clinical utility , especially in the context of distinguishing the difference between utility of a diagnostic test versus test / drug combination versus the drug itself , would be very helpful in dealing with issues of implementation decision and test reimbursement . it should also be noted that even for trastuzumab , which is reimbursed by most insurers , there have been few cost - effectiveness analysis of her2 protein expression and treatment with trastuzumab . for most pharmacogenomic biomarkers , the ideal analyses might not be available until years after the diagnostic test is marketed . with limited comprehensive pharmacoeconomic data for cost - effectiveness evaluations [ 124 , 125 ] , other evaluation approaches ranging from comparing per - patient cost for specific clinical outcome within in - patient setting to decision model - based study that utilizes simulated patient cohort [ 9496 ] should be considered . in addition , all stakeholders should recognize that a negative cost - effectiveness conclusion based primarily on high cost of genotyping needs to be interpreted with the high likelihood of lower cost of genotyping in the foreseeable future . since revenue generation from a pharmacogenomic diagnostic companion test would likely be significantly less than that for a drug , there is not much incentive for pharmaceutical companies to include a thorough cost - effectiveness analysis as part of drug development . with much less financial resources than pharmaceutical companies , the lack of incentive for conducting similar evaluations also applies to diagnostic companies developing the biomarkers . in a way similar to the mutually beneficial codevelopment of proprietary drug and diagnostic test [ 126 , 127 ] , one possible solution is for diagnostic companies to collaborate with other stakeholders , such as pharmacy benefit manager ( pbm ) , to generate the evidence deemed necessary for reimbursement by both private payers and regulatory agencies . medco is the first pbm to use claims data in demonstrating a 28% reduction in bleeding or thromboembolic events in patients whose physicians were provided with cyp2c9 and vkorc1 genotypes results , when compared to patients without genetic testing . concurrent with the clinical effectiveness data is a $ 910 cost saving over a 6-month study period in the genotyped group . this type of economic impact data for pharmacogenomic testing could be used as evidence of cost - effectiveness to insurance payers and administrators of healthcare systems for consideration of potential implementation . given the dilemma of insistence of evidence - based data for reimbursement and the limited financial resource of most diagnostic companies in developing the biomarker , some paradigm shifts in thinking about approaches to reimbursement decision could be offered to the payers . instead of a universal reimbursement for all patients tested for a pharmacogenomic biomarker , an action - based reimbursement could be instituted . using clopidogrel as an example , the differential reimbursement could take the form of no payment for the cyp2c19 genotype test , if no pci is performed and clopidogrel is not prescribed , or even different amount of payment based on the risk of pci . this differential pay concept is currently in place for most prescription drugs in the form of copayment , as well as in coverage amount between within - network versus out - of - network physician visits . adopting such approach would lessen the financial burden for payer since the cost of the one - time test could be easily covered through cost saving associated with not using the drug when it is ineffective or harmful in specific patient populations , and it could provide a work - around to some payers ' insisting on conclusive evidence of linking diagnostic tests to health outcomes . implementation of pharmacogenomic testing could result in situations where an individual 's disease or medical condition is revealed to other parties , however unintended , as well as potential for discrimination and ineligibility for employment and insurance . therefore , even though the public is in general receptive to genetic - based prescribing [ 129 , 130 ] , effort should be directed towards alleviating their concern regarding privacy and confidentiality for the purposes of employment and insurance coverage decisions . they should be informed that there are ways to both protect patients ' privacy whilst at the same time promote the pharmacogenomic implementation in clinical practice [ 131 , 132 ] . in addition , provisions from the 2008 genetic information nondiscrimination act were designed to protect individuals from genetic discrimination . addressing these concerns also encourages informed patients to participate in necessary research [ 115 , 133 ] , for example , comparative effectiveness requested by other stakeholders , as well as facilitate healthcare professionals ' willingness to fully integrate genomic services into clinical practice . despite this , other existing concerns include ownership of genetic materials , availability and access to the information ( both locally and across different health system facilities similar to that of the veterans affairs emr ) , and patient 's awareness of the consequences of storing genetic materials and phenotypic data . these concerns would need to be addressed to the satisfaction of all stakeholders , especially the patients . most discussions and debates on the ethical , legal , and social implications of genetic tests usually make few distinctions between pharmacogenomic biomarkers designed for drug therapy individualization and genetic tests predicting disease susceptibility that usually carry a much greater potential for abuse . for the purpose of implementation , it would seem appropriate that consent for pharmacogenomic biomarker tests designed to individualize their drug therapy ( choice and/or dosage regimen ) not be treated the same extent of scrutiny and requirement as genetic testing for disease susceptibility . a lessening in regulation and consent requirements for pharmacogenomic markers might make it easier for their implementation . however , this issue of is very much open for further discussion before consensus can be made . social concerns also arise from clinical implementation of pharmacogenomic biomarkers within the healthcare systems . in the united states , patients are required to pay for some of the cost of the medical service , either in the form of copayment or coinsurance . therefore , an individual patient 's socioeconomic status could preclude any potential beneficial pharmacogenomic test information and exacerbate health - care disparities among different patients . in addition , for patients who are identified by pharmacogenomic test either as nonresponders or at high risk of adverse drug reaction to a specific drug , the use of pharmacogenomic test as a gatekeeper of accessibility to drug treatment might pose a problem if there is no suitable alternative drug available . as discussed earlier in this paper , carriers of the del allele of the 141c ind / del polymorphism of the dopamine d2 receptor gene are predicted to have poor response to antipsychotic treatment ; yet , all currently marketed antipsychotic treatments possess d2 blockade . is it ethical or appropriate if the patient and/or the physician decide to use a drug regardless of the unfavorable response and/or risk associated with a specific genotype ? these are relevant questions since the clinical validity and clinical utility of most pharmacogenomic tests have not been universally accepted in clinical practice . if a pharmacogenetic test ( e.g. , cyp2c29 ) is used to guide therapy with one drug ( e.g. , warfarin ) and the patient is later prescribed another drug that is also affected by the gene previously tested ( e.g. , phenytoin ) , should the clinician be responsible to act on the genotype results when dosing the second drug ? if the answer is affirmative , then some point - of - care mechanism must be in place , for example , in an emr with pop - up action alert containing the pharmacogenomic information , so that the clinician is aware of genetic test results relevant to the prescribed drug . the immediate implication with availability of pharmacogenomic information within the emr is that the information should not be ignored for clinical , ethical , and legal reasons . pharmacogenomic biomarker tests are a subset of the increasing universe of genetic tests advertised over the internet directly to the consumer . most of these direct - to - consumer ( dtc ) genetic tests are home brew and not subject to regulatory oversight by the fda and/or clia compliance for test quality standards and proficiency . in addition , companies selling dtc genetic tests can develop and market them without establishing clinical utility , which contrasts significantly to that demanded for pharmacogenomic biomarkers discussed earlier in this paper . the lack of regulatory oversight and concern of test validity likely contribute to the conclusion that most dtc genetic tests are not useful in predicting disease risk [ 134 , 135 ] . current knowledge suggests that genomic profiling based on a single snp , a common feature to most dtc genetic tests , is not necessarily clinically accurate or useful . in this regard , the recent report of a dtc genome - wide platform could provide a useful example of the impact of pharmacogenomic profiling on patient care . despite the increased consumer desire for health - related information and personalized medicine , most patients would need the help of clinicians to differentiate the relevance of different pharmacogenomics tests . this underscores the importance of educating clinicians and preparing them to provide the appropriate test interpretation for clinical decision - making . incorporating pharmacogenomics into the entire drug development process holds significant potentials for more efficient and effective clinical trials as well as financial implications for the industry . however , the issues of sufficient sample size , the cost and time associated with conducting a rct to address a specific study hypothesis , and the logistics of ensuring privacy concerns of institutional review board with possible delay in study approval and subject enrollment have posted a significant challenge and deterrent for the industry to fully incorporate pharmacogenomics in different phases of drug development . in addition , the blockbuster drug concept and its financial impact on revenue have historically played a major role in pharmaceutical drug development . as such , the concept of pharmacogenomics and the resultant segmented ( and smaller ) market tailored to a subpopulation with specific genotype however , trastuzumab provides a good example of the benefit of paradigm shift in thinking about market share and revenue . the manufacturer 's development of trastuzumab along with the diagnostic device results in capturing the market share associated with breast cancer drug treatment in all , albeit at a smaller number , of the women overexpressing the her2 protein . there are additional drug development advantages associated with this mental shift in business model from the traditional approach of product differentiation to the new commerce of market segmentation , sometimes even with little or no competition . identifying patients likely to respond to participate in clinical trials could enable benefits to be shown in a smaller number of patients , resulting in more efficient phases ii and iii studies conducted in shorter time frame and reducing the overall cost of drug development . it could also screen out patients likely to have unfavorable side effects that only appear in phase iv postmarketing surveillance studies , and such undesirable events sometime could lead to the inevitable and unfavorable outcomes of postmarketing product recall and litigation . the litigation and financial burden could be further minimized if the pharmaceutical company works with regulatory agencies to incorporate the pharmacogenomic information into a drug label that more accurately describes contraindications , precautions , and warnings . finally , as indicated earlier in this paper , beneficial partnership to develop and market a companion diagnostic test can also lead to additional revenue stream . with more than 50% of new chemical entities failing in expensive phase iii clinical trials , high attrition rate in drug development is a well - known fact for the pharmaceutical industry , and a much less discussed and explored role of pharmacogenomics is the potential of rescuing drugs that fail clinical trials during drug development . the prime example for this benefit is gefitinib , which originally was destined to failure because only a small number of patients with small cell lung cancer responded to the drug . however , in 2004 , published results showed that tumor response to the drug was linked to mutations in her1 . subsequently , development of pharmacogenomic biomarker tests for her1 mutations in patients enables identification of responders for gefitinib [ 139143 ] . this example showed that investigational drugs found to be ineffective or unsafe during phase ii or iii clinical trials might deserve a second look from the perspective of pharmacogenomics . another example is lumiracoxib , a selective cyclooxygenase-2 inhibitor that was withdrawn in 2005 from most global pharmaceutical markets because of hepatotoxicity . recently , singer et al . reported a strong association between patients with hla - dq variant alleles , especially hla - dqa10102 , and elevated transferase levels secondary to lumiracoxib - related liver injury [ 144 , 145 ] . as a result , the manufacturer of lumiracoxib has submitted an application to the ema for its use in targeted subpopulations . therefore , as demonstrated by gefitinib and possibly lumiracoxib , failing drugs can be further developed with a smaller target population with the genetic profile predictive of improved efficacy and/or reduced toxicity . this result can then be used for approval with appropriate product label containing the pharmacogenomic information . in reality , a go - ahead decision by the pharmaceutical company for such drug rescue with potential drug approval is dependent not only on the cost and time associated with developing a companion diagnostic test but also measurable better efficacy than competitor drugs in a smaller number of patients . to facilitate this aspect of drug development , regulatory decision incentives in the form of conditional approval with subsequent requirement of phase iv trial or approval similar to those developed and submitted under the orphan drug act could go a long way to provide sufficient incentive for the pharmaceutical industry . regulatory agencies worldwide , primarily the fda , the ema , and the japanese pharmaceuticals and medical devices agency , have recognized the opportunity to utilize pharmacogenomics in predicting drug response and incorporated pharmacogenomic information into revised labels of approved drugs as well as regulatory review , for example , by the iprg of the fda , that is independent of the drug review itself . nevertheless , relevant drug efficacy and safety data and issues that are important for regulatory decision - making were developed long before the era of pharmacogenomics , and it is unclear how traditional regulatory review would approach the inclusion of any pharmacogenomic data in a new drug application ( nda ) package . as described earlier , the fda has developed multidisciplinary workshop as well as regulatory initiatives such as the voluntary exploratory data submission in the usa , and the pharmacogenomics briefing meetings in europe and japan have attempted to encourage the use and submission of pharmacogenomic data by the pharmaceutical industry . however , concerns and questions remain regarding what type of pharmacogenomic data is necessary and when they should be incorporated in the nda process . although significant scientific and technological advances enable identification of variants in ( or haplotypes linked to ) genes that regulate the disposition and target pathways of drugs , translating the pharmacogenomic findings into clinical practice has been met with continued scientific debates , as well as commercial , economical , educational , ethical , legal , and societal barriers . despite the well - known potentials of improving drug efficacy and safety , as well as the efficiency of the drug development process , the logistical issues and challenges identified for incorporating pharmacogenomics into clinical practice and drug development could only be addressed with all stakeholders in the field working together and occasionally accepting a paradigm change in their current approach .

multiple sclerosis is a chronic demyelinating disease affecting one million people worldwide , with a significant burden of psychiatric comorbidity . depression is the commonest psychiatric manifestation but still remains largely underdiagnosed and undertreated . the present work reviews current knowledge on diagnosis , assessment , and somatic and psychotherapeutic treatment interventions for depression in adult and pediatric populations of patients with multiple sclerosis . the mapping of the human genome and subsequent advancements in genetic technology had provided clinicians and scientists an understanding of the genetic basis of altered drug pharmacokinetics and pharmacodynamics , as well as some examples of applying genomic data in clinical practice . this has raised the public expectation that predicting patients ' responses to drug therapy is now possible in every therapeutic area , and personalized drug therapy would come sooner than later . however , debate continues among most stakeholders involved in drug development and clinical decision - making on whether pharmacogenomic biomarkers should be used in patient assessment , as well as when and in whom to use the biomarker - based diagnostic tests . currently , most would agree that achieving the goal of personalized therapy remains years , if not decades , away . realistic application of genomic findings and technologies in clinical practice and drug development require addressing multiple logistics and challenges that go beyond discovery of gene variants and/or completion of prospective controlled clinical trials . the goal of personalized medicine can only be achieved when all stakeholders in the field work together , with willingness to accept occasional paradigm change in their current approach .

this study combines four coordinated protocols sharing the same dias artificial pancreas technology conducted at the universities of padova ( italy ) and montpellier ( france ) , the university of virginia ( uva ) , and the sansum diabetes research institute , santa barbara , california . to test whether a smart phone is capable of running outpatient closed - loop control , we have configured a system comprising available components , which were linked as follows : cgm idex dias ( running all closed - loop computations , user interface , and communications to peripheral devices ) idex pump . the idex is an experimental device manufactured by insulet ( bedford , ma ) , which combines a dexcom seven plus receiver and omnipod insulin pump . in addition , dias transferred data in real time to a central location allowing remote monitoring of patient state and system functions . the primary engineering end point was the percent time with all system communications working properly ; the protocol criterion for success in this early feasibility study was this time reaching > 80% of the total time of investigation . secondary end points included the estimation of the failure rates of system components , frequency analysis of lost or inaccurate cgm records , and control algorithm performance . the clinical goal was to assess patients and clinicians subjective impressions of the system , i.e. , the feasibility of its ambulatory use , including patient usability and wearability . a total of 20 adults ( age 2165 years ) with type 1 diabetes were studied ( 5 subjects at each site ) . before the tests , a pilot subject was performed in italy , france , and in the united states . all participants were experienced insulin pump users and were required to have the following : prestudy hba1c of 69% ; predefined insulin pump parameters for basal rates , carbohydrate ratios , and insulin sensitivity factors ; and proper mental status / cognition . the exclusion criteria were directed toward safety and included recent history of diabetic ketoacidosis or severe hypoglycemia , pregnancy , breastfeeding , or intention of becoming pregnant ( females ) , uncontrolled arterial hypertension , and conditions that may increase the risk of hypoglycemia or infections . in addition , the united states based studies received food and drug administration approval ( ide # g120032 ) and the european studies received appropriate national - level certifications . all studies were registered with clinicaltrials.gov ( nct01578980 for uva / sansum , nct01447992 for padova , and nct01447979 for montpellier ) . after consent and screening , subjects were trained to use the omnipod insulin pump ( insulet ) and participated in a 3- to 7-day pump initiation if needed . two dexcom seven plus sensors ( dexcom , san diego , ca ) were inserted 2472 h before admission ; throughout the trials , the sensors were calibrated per manufacturer s instructions using commercial glucometers . a calibration was performed before dinner at 7:00 p.m. , thereby allowing for further system - required calibrations to be performed during the timeframes before dinner and before breakfast . per food and drug administration recommendation , an additional ( one - time ) calibration was entered by the study staff if there was a discrepancy in the two sensor readings of 20% or if the cgm was reading < 70 mg / dl and the hemocue value was > 85 mg / dl . participants in italy , france , and virginia stayed at hotels , and participants in california resided at a guest house like outpatient research unit of the sansum consisting of a living room , kitchen , four bedrooms , and bathrooms . the participants in the european studies were admitted individually , one subject at a time ; uva had both single and double admissions ; at sansum , all five subjects were admitted concurrently . subjects checked in by 5:00 p.m. and met with the study team , which confirmed that the subjects had brought their insulin , pump supplies , and regular medications . the subject s pump was removed and the study pump containing the subject s insulin was started . connections were established between dias and one sensor designated as primary ( via the idex ) , the insulin pump ( via the idex ) , and the remote monitoring site . the subject was then introduced to dias operation ; the orientation took 1520 min to complete . the dias user manual ( supplementary data ) and advice from the study team were available to the subjects at all times . after this introduction , the subjects were in charge of their interactions with dias , controlling the system via its graphical user interface . the protocol continued for 42 h. during the first evening / night of study , dias was used in open - loop mode with the subject s home insulin parameters . at 7:00 a.m. on day 2 , the system was switched into closed - loop mode and remained in closed - loop control for 29 h until the subject was discharged at 12:00 p.m. on day 3 . meals were delivered to the patient s room from local restaurants or consumed at local dining facilities ( e.g. , dining out at a restaurant in padova or a hotel buffet breakfast at uva ) . the carbohydrate content of the meals was estimated by the subject and proper entry of the desired carbohydrate amount into dias was confirmed by the study physician , but there were no dietary restrictions . when the subjects were outside of their room , they were accompanied by a member of the study team and dias were remotely monitored continually . figure 1 describes the timeline of the studies in europe and in the united states . protocol design in european ( a ) and united states ( b ) investigation centers . 1b ) were as follows : in padova and montpellier , the patient was moved to the hospital at 7:00 a.m. on day 2 of the study for initiation of closed - loop control and remained in the hospital for 10 h before returning to the hotel for the rest of the study ; in the united states studies the control algorithm was switched into safety - only mode for the night ( 11:00 p.m. to 7:00 a.m. ) , as requested by the food and drug administration . a study physician , a nurse , and a technician were located in nearby rooms to provide assistance if needed . reference blood glucose readings were measured simultaneously by finger stick with a hemocue ( hemocue ab , ngelholm , sweden ) and a commercial glucometer beginning at 7:00 p.m. on the evening of admission and continuing every 2 h during the day . overnight , there were no scheduled finger sticks ; reference blood glucose measurements were taken only if dias or the secondary sensor alarm indicated hypoglycemia or hyperglycemia , or if the two sensors had readings diverging by > 20% . nursing staff checked dias and secondary cgm readings hourly overnight and system alarms were monitored remotely for the dias and with a baby monitor to capture alarms from the secondary cgm . any dias hypoglycemia red - light warning triggered treatment with 15 g fast - acting carbohydrate ( e.g. , juice ) , whereas hyperglycemia red - light warnings prompted checking the insulin pump for occlusion or malfunction . mmol / l ( 250 mg / dl ) was followed by a -hydroxybutyrate test ( finger stick precision xtra -ketone measurement ) ; confirmed -hydroxybutyrate level > 0.6 mmol / l was a criterion for discontinuation of the trial . any hemocue reading < 80 mg / dl was followed - up with additional finger sticks at least every 15 min and any hemocue blood glucose < 70 mg / dl was treated with fast - acting glucose . the hub of the dias system was an off - the - shelf smart phone running the android operating system . to ensure the operation of the smart phone as a medical device , its operating system was modified to disable processes not related to closed - loop control operation and to include self - checks of system integrity . the communications between dias , the idex , and the pump and the sensor were wireless , giving the patient the freedom to be fully detached from the dias controller . the system components worn by the patient included an insulet omnipod insulin pod and a dexcom seven plus sensor / transmitter . the patient additionally wore a pouch containing a communication box ( either viliv s5 tablet or galaxy nexus phone ) attached to the idex . the idex and the communication box were only needed for automated data transfer and pump control at this early feasibility stage . these devices did not have any computing or patient interaction functions and were abandoned in subsequent studies . the subject controlled dias using graphical user interface , which allowed the following : initializing the system with the average daily insulin dose , basal rate , carbohydrate ratio , and correction factor ; displaying cgm traces and insulin delivery graphs ; and real - time interaction , such as entries of sensor calibrations , meal carbohydrate content , premeal capillary glucose level , and other information the subject wished to provide ( e.g. , exercise or hypoglycemia treatment ) . two traffic - light signals presented the degree of risks for hypoglycemia or hyperglycemia as follows : green light , no risks detected ; yellow light , the system is working actively to mitigate the risks by either attenuating insulin delivery if hypoglycemia is anticipated or administering correction insulin if hyperglycemia is predicted ; and red light , which signifies that risks can not be eliminated by adjustment of insulin alone and intervention is required to either consume carbohydrate or ensure that insulin is delivered properly . dias operated in two modes , open - loop ( first 13 h of each study ) controlling the pump per each patient s preset basal / bolus delivery instructions and displaying cgm and insulin delivery information or closed - loop ( hours 1442 of each study ) running a closed - loop control algorithm . both modes of operation included fully automated transfer of data from the sensor to dias and commands from dias to the insulin pump . user input was required only before meals and whenever the system signaled imminent risk for hypoglycemia or hyperglycemia . the closed - loop control algorithm included two modules : 1 ) safety supervision responsible for prediction of hypoglycemia , attenuation , or discontinuation of insulin delivery if hypoglycemia is anticipated and warnings if hypoglycemia is imminent and can not be prevented by insulin discontinuation alone ( 19 ) and 2 ) range - correction module responsible for injecting correction boluses . the clinical use of this algorithm is described in detail in a recent publication as standard control to range ( 12 ) ; details on its engineering architecture also have been published ( 20 ) . occasional cgm data loss ( up to 20 min ) did not stop the operation of the controller ; during loss of pump communication , insulin was not delivered . in addition , dias transmitted data in real time through either 3 g ( telephone network ) or wifi to two servers ( uva and montpellier ) , which allowed team members to log - in for remote observation from their locations . the server connections were one - directional : dias transmitted data out but could not be controlled from a remote location for safety reasons . the transmitted data contained glucose traces , insulin infusion by the pump , and technical information about the functioning of the control algorithm but did not contain any subject identifiers ; the monitoring web sites were password - protected . achieving statistical significance was not an objective of this early - feasibility investigation . the data analysis corresponded to the goals of the study and included estimation of the failure rates of system components , frequency analysis of lost or inaccurate cgm records , and percent time of active system operation . post hoc analyses included t test and nonparametric comparisons of open versus closed - loop parameters of glucose control observed during the study ; however , the study was not powered for this outcome . before inclusion in the analyses , cgm data were sent through retrospective recalibration using reference blood glucose readings as discussed in a recent editorial ( 21 ) . a total of 20 adults ( age 2165 years ) with type 1 diabetes were studied ( 5 subjects at each site ) . before the tests , a pilot subject was performed in italy , france , and in the united states . all participants were experienced insulin pump users and were required to have the following : prestudy hba1c of 69% ; predefined insulin pump parameters for basal rates , carbohydrate ratios , and insulin sensitivity factors ; and proper mental status / cognition . the exclusion criteria were directed toward safety and included recent history of diabetic ketoacidosis or severe hypoglycemia , pregnancy , breastfeeding , or intention of becoming pregnant ( females ) , uncontrolled arterial hypertension , and conditions that may increase the risk of hypoglycemia or infections . all protocols were approved by the review boards of the participating institutions . in addition , the united states based studies received food and drug administration approval ( ide # g120032 ) and the european studies received appropriate national - level certifications . all studies were registered with clinicaltrials.gov ( nct01578980 for uva / sansum , nct01447992 for padova , and nct01447979 for montpellier ) . after consent and screening , subjects were trained to use the omnipod insulin pump ( insulet ) and participated in a 3- to 7-day pump initiation if needed . two dexcom seven plus sensors ( dexcom , san diego , ca ) were inserted 2472 h before admission ; throughout the trials , the sensors were calibrated per manufacturer s instructions using commercial glucometers . a calibration was performed before dinner at 7:00 p.m. , thereby allowing for further system - required calibrations to be performed during the timeframes before dinner and before breakfast . per food and drug administration recommendation , an additional ( one - time ) calibration was entered by the study staff if there was a discrepancy in the two sensor readings of 20% or if the cgm was reading < 70 mg / dl and the hemocue value was > 85 mg / dl . participants in italy , france , and virginia stayed at hotels , and participants in california resided at a guest house like outpatient research unit of the sansum consisting of a living room , kitchen , four bedrooms , and bathrooms . the participants in the european studies were admitted individually , one subject at a time ; uva had both single and double admissions ; at sansum , all five subjects were admitted concurrently . subjects checked in by 5:00 p.m. and met with the study team , which confirmed that the subjects had brought their insulin , pump supplies , and regular medications . the subject s pump was removed and the study pump containing the subject s insulin was started . connections were established between dias and one sensor designated as primary ( via the idex ) , the insulin pump ( via the idex ) , and the remote monitoring site . the subject was then introduced to dias operation ; the orientation took 1520 min to complete . the dias user manual ( supplementary data ) and advice from the study team were available to the subjects at all times . after this introduction , the subjects were in charge of their interactions with dias , controlling the system via its graphical user interface . the protocol continued for 42 h. during the first evening / night of study , dias was used in open - loop mode with the subject s home insulin parameters . at 7:00 a.m. on day 2 , the system was switched into closed - loop mode and remained in closed - loop control for 29 h until the subject was discharged at 12:00 p.m. on day 3 . meals were delivered to the patient s room from local restaurants or consumed at local dining facilities ( e.g. , dining out at a restaurant in padova or a hotel buffet breakfast at uva ) . the carbohydrate content of the meals was estimated by the subject and proper entry of the desired carbohydrate amount into dias was confirmed by the study physician , but there were no dietary restrictions . when the subjects were outside of their room , they were accompanied by a member of the study team and dias were remotely monitored continually . figure 1 describes the timeline of the studies in europe and in the united states . protocol design in european ( a ) and united states ( b ) investigation centers . 1b ) were as follows : in padova and montpellier , the patient was moved to the hospital at 7:00 a.m. on day 2 of the study for initiation of closed - loop control and remained in the hospital for 10 h before returning to the hotel for the rest of the study ; in the united states studies the control algorithm was switched into safety - only mode for the night ( 11:00 p.m. to 7:00 a.m. ) , as requested by the food and drug administration . a study physician , a nurse , and a technician were located in nearby rooms to provide assistance if needed . reference blood glucose readings were measured simultaneously by finger stick with a hemocue ( hemocue ab , ngelholm , sweden ) and a commercial glucometer beginning at 7:00 p.m. on the evening of admission and continuing every 2 h during the day . overnight , there were no scheduled finger sticks ; reference blood glucose measurements were taken only if dias or the secondary sensor alarm indicated hypoglycemia or hyperglycemia , or if the two sensors had readings diverging by > 20% . nursing staff checked dias and secondary cgm readings hourly overnight and system alarms were monitored remotely for the dias and with a baby monitor to capture alarms from the secondary cgm . any dias hypoglycemia red - light warning triggered treatment with 15 g fast - acting carbohydrate ( e.g. , juice ) , whereas hyperglycemia red - light warnings prompted checking the insulin pump for occlusion or malfunction . mmol / l ( 250 mg / dl ) was followed by a -hydroxybutyrate test ( finger stick precision xtra -ketone measurement ) ; confirmed -hydroxybutyrate level > 0.6 mmol / l was a criterion for discontinuation of the trial . any hemocue reading < 80 mg / dl was followed - up with additional finger sticks at least every 15 min and any hemocue blood glucose < 70 mg / dl was treated with fast - acting glucose . the hub of the dias system was an off - the - shelf smart phone running the android operating system . to ensure the operation of the smart phone as a medical device , its operating system was modified to disable processes not related to closed - loop control operation and to include self - checks of system integrity . the communications between dias , the idex , and the pump and the sensor were wireless , giving the patient the freedom to be fully detached from the dias controller . the system components worn by the patient included an insulet omnipod insulin pod and a dexcom seven plus sensor / transmitter . the patient additionally wore a pouch containing a communication box ( either viliv s5 tablet or galaxy nexus phone ) attached to the idex . the idex and the communication box were only needed for automated data transfer and pump control at this early feasibility stage . these devices did not have any computing or patient interaction functions and were abandoned in subsequent studies . the subject controlled dias using graphical user interface , which allowed the following : initializing the system with the average daily insulin dose , basal rate , carbohydrate ratio , and correction factor ; displaying cgm traces and insulin delivery graphs ; and real - time interaction , such as entries of sensor calibrations , meal carbohydrate content , premeal capillary glucose level , and other information the subject wished to provide ( e.g. , exercise or hypoglycemia treatment ) . two traffic - light signals presented the degree of risks for hypoglycemia or hyperglycemia as follows : green light , no risks detected ; yellow light , the system is working actively to mitigate the risks by either attenuating insulin delivery if hypoglycemia is anticipated or administering correction insulin if hyperglycemia is predicted ; and red light , which signifies that risks can not be eliminated by adjustment of insulin alone and intervention is required to either consume carbohydrate or ensure that insulin is delivered properly . dias operated in two modes , open - loop ( first 13 h of each study ) controlling the pump per each patient s preset basal / bolus delivery instructions and displaying cgm and insulin delivery information or closed - loop ( hours 1442 of each study ) running a closed - loop control algorithm . both modes of operation included fully automated transfer of data from the sensor to dias and commands from dias to the insulin pump . user input was required only before meals and whenever the system signaled imminent risk for hypoglycemia or hyperglycemia . the closed - loop control algorithm included two modules : 1 ) safety supervision responsible for prediction of hypoglycemia , attenuation , or discontinuation of insulin delivery if hypoglycemia is anticipated and warnings if hypoglycemia is imminent and can not be prevented by insulin discontinuation alone ( 19 ) and 2 ) range - correction module responsible for injecting correction boluses . the clinical use of this algorithm is described in detail in a recent publication as standard control to range ( 12 ) ; details on its engineering architecture also have been published ( 20 ) . occasional cgm data loss ( up to 20 min ) did not stop the operation of the controller ; during loss of pump communication , insulin was not delivered . in addition , dias transmitted data in real time through either 3 g ( telephone network ) or wifi to two servers ( uva and montpellier ) , which allowed team members to log - in for remote observation from their locations . the server connections were one - directional : dias transmitted data out but could not be controlled from a remote location for safety reasons . the transmitted data contained glucose traces , insulin infusion by the pump , and technical information about the functioning of the control algorithm but did not contain any subject identifiers ; the monitoring web sites were password - protected . the data analysis corresponded to the goals of the study and included estimation of the failure rates of system components , frequency analysis of lost or inaccurate cgm records , and percent time of active system operation . post hoc analyses included t test and nonparametric comparisons of open versus closed - loop parameters of glucose control observed during the study ; however , the study was not powered for this outcome . before inclusion in the analyses , cgm data were sent through retrospective recalibration using reference blood glucose readings as discussed in a recent editorial ( 21 ) . the focus of this investigation was on the concept of using dias as a smart phone based control algorithm and user interface host . we assessed dias in terms of human factors and usability , system and component performance , performance of the control algorithm , utility of remote monitoring , and clinical events . before this study , a formative evaluation of the dias user interface heuristic evaluation ( expert review ) was followed by three focus groups with type 1 diabetic patients with varying exposure to diabetes technology ( n = 13 ) . feedback was gathered on various system components addressing user interaction , system features , and capabilities . change recommendations were prioritized , and users were asked to rate the system on several criteria . users indicated the importance of maintaining all existing insulin pump and cgm device functionalities ( 22 ) . the dias graphical user interface ( supplementary data ) proved to be reliable and well - understood by the subjects . all were able to easily navigate through the graphical user interface commands on their own in both open - loop and closed - loop modes of operation , view cgm and insulin information , and administer meal or correction boluses as needed . one subject used a hotel treadmill , one subject in italy rode a bike , one subject in france walked to nearby museums , and five subjects took a shower with the pouch hanging just outside of the shower . system wearability was evaluated in relative terms , comparing dias to previous laptop - based systems . with the transition to a smart phone as a system hub and to wireless data transmission , the weight of a closed - loop control system was reduced several - fold . figure 2a presents photos of dias displaying cgm and insulin delivery traces and the entire system worn by a study subject . dias communicated wirelessly with the idex / communication box ; these devices are placed in a pouch on the patient s belt . the idex communicated wirelessly to an omnipod insulin pump and to a dexcom sensor visible as attached on the subject . the communication range of the idex with the insulin pod and dexcom sensor was 5 inches , which necessitated the use of a belt pouch . with this set - up , the subjects were able to maintain activities of daily living , a necessary first step toward routine outpatient use . a : photos of the dias smart phone displaying cgm and insulin delivery traces ( left ) and the entire system worn by a study subject ( right ) . each of the five subjects participating simultaneously in these trials is represented by an icon on the computer screen . table 1 presents metrics of the technical performance of the artificial pancreas system overall and during the open - loop and closed - loop portions of the study . two subjects described developed hyperglycemia with ketones because of pump site or pod failures in the initial open - loop portion of the study and were rescheduled . only the completed second study data for the rescheduled subjects are included in this analysis . additionally , the three pilot subjects for each country were not included in the analysis ; the data of the first two from italy and france were recently published ( 18 ) . overall , the artificial pancreas system was functional 98% of the time , which exceeded the initially set primary end point goal of 80% . performance metrics for the functioning of the artificial pancreas system used in these studies and of its primary components one element of system connectivity should be noted . in the european studies and in the first united states based studies we used a viliv s5 tablet to communicate with the idex , which was then replaced by a more reliable samsung galaxy nexus smart phone . as evident from table 1 , this replacement had a substantial effect on system reliability , reducing almost five - fold the number of unplanned system restarts because of loss of signal transmission . because the communication box was dedicated solely to data transmission , its replacement did not affect the conceptual or the computing outcomes of the study . further , table 1 presents data on the performance of the principal system components : the cgm , dias , and the insulin pump . of particular importance for fully integrated closed - loop control is the stability of interdevice connections ( sensor idex smart phone idex insulin pump ) . table 1 presents the availability of cgm and insulin pump communications with dias during the study . although the study was not designed to test algorithm performance or to compare open - loop versus closed - loop , table 2 presents a set of glycemic control metrics and certain post hoc comparisons of open - loop versus closed - loop nights using retrospectively recalibrated cgm data ( 21 ) . the outpatient performance of the controller was similar to its inpatient performance of this same control algorithm observed in a previous study ( standard control to range , 12 ) ; thus , first indications are that a different platform ( e.g. , a smart phone ) under different outpatient conditions may achieve similar performance as a laptop - based system in the hospital . on open - loop versus closed - loop control , we observed 80 vs.72% time within target range ( p = 0.22 ) and 0.53 vs. 0.27 hypoglycemic episodes 3.9 mmol / l ( 70 mg / dl ) per 24 h ( p = 0.16 ) ; in other words , there were no significant differences between open - loop and closed - loop control overnight , which is an expected result for standard control to range ( 12 ) . performance of the control algorithm figure 2b presents a screenshot of the remote monitoring system operation during the study at sansum . each of the five subjects participating simultaneously in this study is represented by an icon on the computer screen . the icon summarizes real - time information , including patient identification number , current cgm reading and direction of change , the state of the hypoglycemia and hyperglycemia alerts , and a message informing the technician of possible risks or system malfunction . safety supervision module was active for three patients ( identification numbers 211 , 212 , and 214 ) as indicated by yellow hypoglycemia lights . each icon can be clicked during a monitoring session , which will display more detailed information for this subject , including detailed records of insulin delivery , glucose data , and the algorithm functions . throughout the study , observation of the participants was performed mainly through the remote monitoring system , which proved to be a useful tool . on six occasions during the study ( two during open - loop and four during closed - loop control ) , carbohydrate treatment was administered for blood glucose levels < 3.3 mmol / l ( 60 mg / dl ) , for a total of 0.17 events per 24 h of system operation . there were no instances of patient - initiated system shutdown , but the trials were discontinued by study staff on three occasions . the first two events occurred early , before initiation of closed - loop control , and the subjects were rescheduled for subsequent admissions , which concluded successfully . subject 1 experienced hyperglycemia of 260 mg / dl with -hydroxybutyrate level of 0.7 mmol / l 2 h after the insulin pump was initiated ( consistent with pod compared with insertion site failure ) . after the connection was reestablished with a new tablet , the insulin pod alarmed , prompting a pod change . the new pod occluded ( blood noted in pod tubing ) , resulting in -hydroxybutyrate of 1.3 mmol / l . 1 ) with the range controller switched off , delivering only basal rate ( 4.1 units in the previous 6 h ) . the subject s cgm glucose was noted to increase from 180 mg / dl to 295 mg / dl over the final 2 h , suggesting that a pod occlusion ( unconfirmed ) may have contributed to this event . these three patients were treated with subcutaneous insulin , resulting in prompt resolution of the mild ketosis . before this study , a formative evaluation of the dias user interface was conducted to evaluate the feasibility of the design for patient use . heuristic evaluation ( expert review ) was followed by three focus groups with type 1 diabetic patients with varying exposure to diabetes technology ( n = 13 ) . feedback was gathered on various system components addressing user interaction , system features , and capabilities . change recommendations were prioritized , and users were asked to rate the system on several criteria . users indicated the importance of maintaining all existing insulin pump and cgm device functionalities ( 22 ) . the dias graphical user interface ( supplementary data ) proved to be reliable and well - understood by the subjects . all were able to easily navigate through the graphical user interface commands on their own in both open - loop and closed - loop modes of operation , view cgm and insulin information , and administer meal or correction boluses as needed . one subject used a hotel treadmill , one subject in italy rode a bike , one subject in france walked to nearby museums , and five subjects took a shower with the pouch hanging just outside of the shower . system wearability was evaluated in relative terms , comparing dias to previous laptop - based systems . with the transition to a smart phone as a system hub and to wireless data transmission , figure 2a presents photos of dias displaying cgm and insulin delivery traces and the entire system worn by a study subject . dias communicated wirelessly with the idex / communication box ; these devices are placed in a pouch on the patient s belt . the idex communicated wirelessly to an omnipod insulin pump and to a dexcom sensor visible as attached on the subject . the communication range of the idex with the insulin pod and dexcom sensor was 5 inches , which necessitated the use of a belt pouch . with this set - up , the subjects were able to maintain activities of daily living , a necessary first step toward routine outpatient use . a : photos of the dias smart phone displaying cgm and insulin delivery traces ( left ) and the entire system worn by a study subject ( right ) . each of the five subjects participating simultaneously in these trials is represented by an icon on the computer screen . table 1 presents metrics of the technical performance of the artificial pancreas system overall and during the open - loop and closed - loop portions of the study . two subjects described developed hyperglycemia with ketones because of pump site or pod failures in the initial open - loop portion of the study and were rescheduled . only the completed second study data for the rescheduled subjects are included in this analysis . additionally , the three pilot subjects for each country were not included in the analysis ; the data of the first two from italy and france were recently published ( 18 ) . overall , the artificial pancreas system was functional 98% of the time , which exceeded the initially set primary end point goal of 80% . performance metrics for the functioning of the artificial pancreas system used in these studies and of its primary components one element of system connectivity should be noted . in the european studies and in the first united states based studies we used a viliv s5 tablet to communicate with the idex , which was then replaced by a more reliable samsung galaxy nexus smart phone . as evident from table 1 , this replacement had a substantial effect on system reliability , reducing almost five - fold the number of unplanned system restarts because of loss of signal transmission . because the communication box was dedicated solely to data transmission , its replacement did not affect the conceptual or the computing outcomes of the study . further , table 1 presents data on the performance of the principal system components : the cgm , dias , and the insulin pump . of particular importance for fully integrated closed - loop control is the stability of interdevice connections ( sensor idex smart phone idex insulin pump ) . table 1 presents the availability of cgm and insulin pump communications with dias during the study . although the study was not designed to test algorithm performance or to compare open - loop versus closed - loop , table 2 presents a set of glycemic control metrics and certain post hoc comparisons of open - loop versus closed - loop nights using retrospectively recalibrated cgm data ( 21 ) . the outpatient performance of the controller was similar to its inpatient performance of this same control algorithm observed in a previous study ( standard control to range , 12 ) ; thus , first indications are that a different platform ( e.g. , a smart phone ) under different outpatient conditions may achieve similar performance as a laptop - based system in the hospital . on open - loop versus closed - loop control , we observed 80 vs.72% time within target range ( p = 0.22 ) and 0.53 vs. 0.27 hypoglycemic episodes 3.9 mmol / l ( 70 mg / dl ) per 24 h ( p = 0.16 ) ; in other words , there were no significant differences between open - loop and closed - loop control overnight , which is an expected result for standard control to range ( 12 ) . figure 2b presents a screenshot of the remote monitoring system operation during the study at sansum . each of the five subjects participating simultaneously in this study is represented by an icon on the computer screen . the icon summarizes real - time information , including patient identification number , current cgm reading and direction of change , the state of the hypoglycemia and hyperglycemia alerts , and a message informing the technician of possible risks or system malfunction . safety supervision module was active for three patients ( identification numbers 211 , 212 , and 214 ) as indicated by yellow hypoglycemia lights . each icon can be clicked during a monitoring session , which will display more detailed information for this subject , including detailed records of insulin delivery , glucose data , and the algorithm functions . throughout the study , observation of the participants was performed mainly through the remote monitoring system , which proved to be a useful tool . on six occasions during the study ( two during open - loop and four during closed - loop control ) , carbohydrate treatment was administered for blood glucose levels < 3.3 mmol / l ( 60 mg / dl ) , for a total of 0.17 events per 24 h of system operation . there were no instances of patient - initiated system shutdown , but the trials were discontinued by study staff on three occasions . the first two events occurred early , before initiation of closed - loop control , and the subjects were rescheduled for subsequent admissions , which concluded successfully . subject 1 experienced hyperglycemia of 260 mg / dl with -hydroxybutyrate level of 0.7 mmol / l 2 h after the insulin pump was initiated ( consistent with pod compared with insertion site failure ) . after the connection was reestablished with a new tablet , the insulin pod alarmed , prompting a pod change . the new pod occluded ( blood noted in pod tubing ) , resulting in -hydroxybutyrate of 1.3 mmol / l . 1 ) with the range controller switched off , delivering only basal rate ( 4.1 units in the previous 6 h ) . the subject s cgm glucose was noted to increase from 180 mg / dl to 295 mg / dl over the final 2 h , suggesting that a pod occlusion ( unconfirmed ) may have contributed to this event . these three patients were treated with subcutaneous insulin , resulting in prompt resolution of the mild ketosis . technology advancements in the past year made possible the development of dias , wearable ambulatory artificial pancreas platforms using an off - the - shelf smart phone as a computational hub . besides more user - friendly touch - screen interface and wireless connectivity , one easily quantifiable result of the transition from a laptop - based to a phone - based closed - loop control is a significant reduction in the system weight , which brings the system one important step closer to ambulatory use . ultimately , this would lead to closing the loop with a portable minimally invasive system suitable for home use . industry is currently transitioning cgms and pumps to include wireless connectivity ; thus , dias is only the first of many portable devices that will be capable of wireless data exchange and fully integrated closed - loop control . at the time of this outpatient trial , the dexcom seven plus and the omnipod insulet pump had short - range wireless capability to communicate with an idex research platform . also , for the idex to establish wireless communication with dias , an intermediary tablet ( or a cell phone ) needed to be connected to the idex . hence , there was short - range wireless communication from the patient ( wearing a pod and sensor / transmitter ) to a pouch containing the idex and tablet ( or cell phone ) , and long - range wireless communication between the pouch and the dias artificial pancreas platform . these intermediate devices are now being phased out ; communication boxes are no longer necessary . such a technology improvement was anticipated in our study ; thus , we focused on the smart phone computing and user - interface capabilities of the dias , assuming that this would be the device that is here to stay . special emphasis should be placed on the fact that the subjects were operating the system by themselves most of the time . to the best of our knowledge , this is the first trial in which the subjects were responsible for the oversight of their closed - loop systems , a step that is critical for outpatient deployment of closed - loop control . based on this feedback , we conclude that the form factor of dias as an artificial pancreas platform is appropriate for outpatient use . however , before long - term efficacy studies comparing outpatient artificial pancreas with sensor - augmented pump therapy can proceed , system wearability during daily living and the reliability of device communications must be ensured . testing of the system at four different sites in three countries and in a variety of hotel and restaurant settings using one , two , or five systems concurrently provided an opportunity to challenge dias with multiple scenarios that are likely to be encountered in nonhospital and , ultimately , home settings . in general , the technical performance of the dias system with overall operational time of 98% exceeded the set goal of 80% . in retrospect , this goal may have been conservative , but before this study it was generally unclear whether a smart phone can run closed - loop control , and there was no experience to guide the choice of this goal . the principal system components sensor , dias , and insulin pump were reasonably reliable , with 0.03 , 0.09 , and 0.12 malfunction events necessitating device replacement per 24 h , respectively . occasional cgm data points were lost ( 8.1% ) , but this did not result in skipping control cycles or discontinuation of the study ; by design , control to range would function during transient absence of cgm data because the controller intervenes only if risks for hypoglycemia or hyperglycemia are detected ( 20 ) . finally , we must emphasize the utility of remote monitoring , which was available on site and at remote locations ( i.e. , studies in europe or in california were observed from virginia and vice versa in real time ) . this was a critical aspect for patient safety that allowed close supervision so that intervention could occur quickly if needed . our system allowed monitoring concurrently multiple patients , a feature that was tested at sansum with five patients simultaneously . this feature alone will allow acceleration of the number of subjects who could be studied at the same time , reducing staffing costs and making artificial pancreas research more efficient . in summary , a wearable inexpensive closed - loop control platform ( dias ) combined with real - time remote monitoring , this system opens the possibility for large pivotal trials that will establish the artificial pancreas as a viable mainstream treatment strategy in type 1 diabetes . participants for this study were recruited from two urban areas ( yaound , the capital city of cameroon , and bamenda , the capital city of the north western region ) and two rural areas ( mbankomo and bafut ) of cameroon . the inhabitants of the urban sites are mainly middle - class public or private sector workers or businessmen . residential houses in the urban areas are grouped closely together and arranged in blocks with paved or graded road access . in the rural areas , the rural habitation pattern is sparse , with most of the homes and farms accessed only via foot paths . as a result of the absence of complete population registers , a sampling frame was established following enumeration of eligible adults in houses in delimited areas of the study sites . eligible participants were adults aged between 25 and 55 years without previously diagnosed diabetes or any known cardiovascular disease . all 3,854 eligible participants ( rural : n = 2,238 , mean age 35.3 7.7 years ; urban : n = 1,616 , mean age 35.4 8.3 years ) in the sampling frame were approached through door - to - door recruitment and invited to participate in the study . a total of 651 volunteers ( rural : n = 303 , mean age 38.5 8.3 years ; urban : n = 348 , mean age 37.9 9.1 years ) took part in this study . volunteers were provided detailed information about the study verbally and in writing , and invited to attend a testing session at their local hospital . volunteers were asked to refrain from eating , drinking or smoking starting at 10:00 p.m. the evening prior to testing . personal and sociodemographic data , as well as data on smoking , alcohol consumption , and fruit and vegetable intake were collected by self - report using an adapted world health organization ( who ) stepwise approach to sureveillance questionnaire . ethical approval for the study was obtained from the cameroon national ethics committee , and all participants provided signed informed consent . pregnant women and other volunteers who could not take part in exercise testing according to study protocol were excluded from the study . an exercise screening questionnaire , adapted from the who rose angina questionnaire was used to screen for preexisting cardiovascular disease which would contraindicate exercise testing . free - living physical activity was measured using a combined heart rate ( hr ) and movement sensor ( actiheart ; cambridge neurotechnology , cambridge , u.k . ) which has been validated in this population in free - living adults with doubly labeled water method as criterion ( 14 ) . the individual hr response to the mechanical work during an 8-min incremental step test was used for individual calibration of hr data ( 15 ) . during the test , hr was monitored in real time and the test was aborted if hr reached 90% of the age - predicted maximum hr ( 16 ) , if the subject could not keep up with the stepping protocol , or if they requested to stop . free - living physical activity was measured in 1-min epochs using the combined hr and movement sensor over 7 continuous days . the participants were requested to carry on with their normal habitual lifestyle and wear the monitor at all times except while showering , bathing , or swimming . participants with less than 2 full days of free - living data ( n = 51 ) were not included in these analyses . hr data were preprocessed ( 17 ) and converted to energy expenditure using the individual calibration derived from the step test , and the accelerometer data were converted to energy expenditure using group acceleration equations corresponding to level walking or running ( 15 ) . minute - by - minute hr and movement - derived physical activity intensity was combined in a branched equation model ( 18 ) to calculate free - living paee . basal metabolic rate ( ~resting energy expenditure [ ree ] ) was estimated using the oxford equations ( 19 ) , and total energy expenditure ( tee ) was calculated by adding ree and a component for the thermic effect of food ( equivalent to 10% of tee ) to the derived paee . energy expenditure variables are expressed per kilogram body weight to adjust for differences in body size . the distribution of intensity of activity was expressed as average daily time ( min / day ) spent at 1 met equal to time spent asleep or sedentary , between 1 and 3 mets equal to light physical activity , above 3 mets equal to moderate - to - vigorous physical activity . an estimate of cardiorespiratory fitness ( vo2max in ml of o2/kg / min ) was calculated as the intensity of work at the age - predicted maximum heart rate ( 15 ) by extrapolation of the individual heart rate response to mechanical work derived from the step test calibration . body weight was measured using electronic clinical scales , and body composition was measured using bio - impedance ( tanita tbf-531 scales ; tanita uk , uxbridge , middlesex , u.k . ) . bmi was calculated as the ratio of body weight ( kg ) to the square of height ( m ) and categorized as normal : < 25 ; overweight : 25 to < 30 ; and obese : 30 waist circumference was measured at the level of the midpoint between the lower costal margin and the anterior superior iliac crests . measurement was made to the nearest 0.1 cm using a nonstretch fiberglass tape measure with participants wearing light indoor clothing . central obesity was defined as waist circumference 80 cm in women or 94 cm in men , or using the cut points of the national cholesterol education program - adult treatment panel iii ( ncep - atp iii ) ( 20 ) when included to define the metabolic syndrome . measurements were made on the dominant arm after 5 min of rest with the subject seated . three measurements were taken at 1-min intervals , and the average of the three constituted the final blood pressure value . capillary blood glucose readings were measured after an overnight fast of at least 8 h , and then 2 h after ingestion of 75 g of glucose dissolved in 300 ml of water . blood glucose measurements were done on whole fresh capillary blood using a hemocue b - glucose analyzer ( hemocue ab , ngelholm , sweden ) . the metabolic syndrome was defined using the ncep - atp iii criteria ( 20 ) . to demonstrate the population distribution of global metabolic health , we computed a continuous summary metabolic risk score ( z - score ) largely on the basis of the components of the metabolic syndrome . this variable was derived by standardizing and then summing waist circumference , blood pressure ( average of systolic and diastolic ) , blood glucose ( average of fasting , 30-min , and 2-h glucose ) , triglycerides , body fat , fasting insulin , and inverse hdl cholesterol . the standardization of these continuous variables was computed by subtracting the sample mean from the individual value and then dividing by the sd of the sample mean . sex - specific calculations were made for waist circumference , body fat , fasting insulin , and hdl cholesterol . fasting blood samples were collected in the mornings between 7:30 and 9:30 a.m. plasma and serum samples were separated upon collection in a refrigerated centrifuge at 4c . samples were transported on dry ice by air to cambridge ( u.k . ) and stored at 80c until further analyses . assays were performed by the national institute for health research cambridge biomedical research centre , core biochemical assay laboratory . hdl cholesterol and triglycerides were measured using automated assays on the dade behring dimension rxl analyzer ( siemens healthcare , camberley , u.k . ) . hdl was measured using a homogenous accelerator selective detergent assay and triglycerides were measured using an enzymatic assay according to manufacturers specifications . ldl was calculated by the friedwald formula ( ldl = cholesterol hdl [ triglycerides/2.2 ] ) . fasting insulin was assayed in singleton on a 1235 autodelfia automatic immunoassay system using a two - step time resolved fluorometric assay kit manufactured by perkin elmer life sciences ( wallac oy , turku , finland ) . pmol / l , 3233 split proinsulin 1% at 2,800 pmol / l , and c - peptide < 0.1% at 5,280 analyses were carried out using stata version 10.1 ( statacorp , college station , tx ) . descriptive characteristics of the study sample are presented as means with sd , adjusted means with se , or as numbers with proportions , stratified by sex - specific paee quartiles . for dichotomous grouping variables , difference in means was assessed using the student t test and difference in proportion using the test or fisher exact test . independent associations between paee and the metabolic syndrome were examined in multivariate logistic regression models . there was no paee interaction by residential site or sex on its association with the metabolic syndrome . models were either unadjusted or adjusted for age , sex , residential site , smoking , alcohol consumption , fruit and vegetable consumption , and educational level ( as a surrogate for socioeconomic status ) . to illustrate the population impact of physical inactivity on metabolic health , we calculated the population attributable fraction of prevalent metabolic syndrome due to being in the lowest quartile of paee ( q1-paee ) . the population attributable fraction was computed as = ( prevalence of q1-paee in case subjects prevalence of q1-paee in control subjects)/(1 prevalence of q1-paee in control subjects ) . free - living physical activity was measured using a combined heart rate ( hr ) and movement sensor ( actiheart ; cambridge neurotechnology , cambridge , u.k . ) which has been validated in this population in free - living adults with doubly labeled water method as criterion ( 14 ) . the individual hr response to the mechanical work during an 8-min incremental step test was used for individual calibration of hr data ( 15 ) . during the test , hr was monitored in real time and the test was aborted if hr reached 90% of the age - predicted maximum hr ( 16 ) , if the subject could not keep up with the stepping protocol , or if they requested to stop . free - living physical activity was measured in 1-min epochs using the combined hr and movement sensor over 7 continuous days . the participants were requested to carry on with their normal habitual lifestyle and wear the monitor at all times except while showering , bathing , or swimming . participants with less than 2 full days of free - living data ( n = 51 ) were not included in these analyses . hr data were preprocessed ( 17 ) and converted to energy expenditure using the individual calibration derived from the step test , and the accelerometer data were converted to energy expenditure using group acceleration equations corresponding to level walking or running ( 15 ) . minute - by - minute hr and movement - derived physical activity intensity was combined in a branched equation model ( 18 ) to calculate free - living paee . basal metabolic rate ( ~resting energy expenditure [ ree ] ) was estimated using the oxford equations ( 19 ) , and total energy expenditure ( tee ) was calculated by adding ree and a component for the thermic effect of food ( equivalent to 10% of tee ) to the derived paee . energy expenditure variables are expressed per kilogram body weight to adjust for differences in body size . the distribution of intensity of activity was expressed as average daily time ( min / day ) spent at 1 met equal to time spent asleep or sedentary , between 1 and 3 mets equal to light physical activity , above 3 mets equal to moderate - to - vigorous physical activity . an estimate of cardiorespiratory fitness ( vo2max in ml of o2/kg / min ) was calculated as the intensity of work at the age - predicted maximum heart rate ( 15 ) by extrapolation of the individual heart rate response to mechanical work derived from the step test calibration . body weight was measured using electronic clinical scales , and body composition was measured using bio - impedance ( tanita tbf-531 scales ; tanita uk , uxbridge , middlesex , u.k . ) . bmi was calculated as the ratio of body weight ( kg ) to the square of height ( m ) and categorized as normal : < 25 ; overweight : 25 to < 30 ; and obese : 30 waist circumference was measured at the level of the midpoint between the lower costal margin and the anterior superior iliac crests . measurement was made to the nearest 0.1 cm using a nonstretch fiberglass tape measure with participants wearing light indoor clothing . central obesity was defined as waist circumference 80 cm in women or 94 cm in men , or using the cut points of the national cholesterol education program - adult treatment panel iii ( ncep - atp iii ) ( 20 ) when included to define the metabolic syndrome . measurements were made on the dominant arm after 5 min of rest with the subject seated . three measurements were taken at 1-min intervals , and the average of the three constituted the final blood pressure value . capillary blood glucose readings were measured after an overnight fast of at least 8 h , and then 2 h after ingestion of 75 g of glucose dissolved in 300 ml of water . blood glucose measurements were done on whole fresh capillary blood using a hemocue b - glucose analyzer ( hemocue ab , ngelholm , sweden ) . the metabolic syndrome was defined using the ncep - atp iii criteria ( 20 ) . to demonstrate the population distribution of global metabolic health , we computed a continuous summary metabolic risk score ( z - score ) largely on the basis of the components of the metabolic syndrome . this variable was derived by standardizing and then summing waist circumference , blood pressure ( average of systolic and diastolic ) , blood glucose ( average of fasting , 30-min , and 2-h glucose ) , triglycerides , body fat , fasting insulin , and inverse hdl cholesterol . the standardization of these continuous variables was computed by subtracting the sample mean from the individual value and then dividing by the sd of the sample mean . sex - specific calculations were made for waist circumference , body fat , fasting insulin , and hdl cholesterol . fasting blood samples were collected in the mornings between 7:30 and 9:30 a.m. plasma and serum samples were separated upon collection in a refrigerated centrifuge at 4c . samples were transported on dry ice by air to cambridge ( u.k . ) and stored at 80c until further analyses . assays were performed by the national institute for health research cambridge biomedical research centre , core biochemical assay laboratory . hdl cholesterol and triglycerides were measured using automated assays on the dade behring dimension rxl analyzer ( siemens healthcare , camberley , u.k . ) . hdl was measured using a homogenous accelerator selective detergent assay and triglycerides were measured using an enzymatic assay according to manufacturers specifications . ldl was calculated by the friedwald formula ( ldl = cholesterol hdl [ triglycerides/2.2 ] ) . fasting insulin was assayed in singleton on a 1235 autodelfia automatic immunoassay system using a two - step time resolved fluorometric assay kit manufactured by perkin elmer life sciences ( wallac oy , turku , finland ) . pmol / l , 3233 split proinsulin 1% at 2,800 pmol / l , and c - peptide < 0.1% at 5,280 analyses were carried out using stata version 10.1 ( statacorp , college station , tx ) . descriptive characteristics of the study sample are presented as means with sd , adjusted means with se , or as numbers with proportions , stratified by sex - specific paee quartiles . for dichotomous grouping variables , difference in means was assessed using the student t test and difference in proportion using the test or fisher exact test . independent associations between paee and the metabolic syndrome were examined in multivariate logistic regression models . there was no paee interaction by residential site or sex on its association with the metabolic syndrome . models were either unadjusted or adjusted for age , sex , residential site , smoking , alcohol consumption , fruit and vegetable consumption , and educational level ( as a surrogate for socioeconomic status ) . to illustrate the population impact of physical inactivity on metabolic health , we calculated the population attributable fraction of prevalent metabolic syndrome due to being in the lowest quartile of paee ( q1-paee ) . the population attributable fraction was computed as = ( prevalence of q1-paee in case subjects prevalence of q1-paee in control subjects)/(1 prevalence of q1-paee in control subjects ) . descriptive characteristics of the study sample stratified by sex and rural or urban residence are presented in supplementary table 1 . clinical and metabolic characteristics of the study sample stratified by sex - specific quartiles of paee are presented in table 1 . most of the individual metabolic and clinical parameters , as well as the clustered metabolic risk score or prevalence of the metabolic syndrome showed a significant linear trend across quartiles of paee . blood lipids were generally low in this sample with no sex or rural - urban differences . prevalence of metabolic syndrome was almost fourfold lower in the highest compared with the lowest quartile of paee . clinical and metabolic characteristics by quartiles of paee in adult cameroonians ( n = 552 ) data are age - adjusted means se or n ( % ) . bg , blood glucose ; bp , blood pressure ; ms , metabolic syndrome ; wc , waist circumference ; zms , clustered metabolic risk score . urban dwellers had a significantly lower paee than rural dwellers ( 44.2 21.0 vs. 59.6 23.7 kj / kg / day , p < 0.001 ) and a higher prevalence of the metabolic syndrome ( 17.7 vs. 3.5% , p < 0.001 ) ( supplementary tables 1 and 2 ) . there was no significant difference between the sexes in metabolic risk expressed as a continuous clustered metabolic score , but urban dwellers had a significantly higher mean score than rural dwellers ( 0.80 3.67 vs. 1.11 2.90 , p < 0.001 ) . this difference was observed to correspond to a rural - to - urban right shift in the population density distribution of the clustered metabolic z - score when stratified by residential area ( fig . a similar but reverse difference was observed with the population distribution of paee , with a rural - to - urban left shift in the distribution . population distribution of paee and clustered metabolic z - score in adults in rural and urban cameroon . table 2 presents independent associations between paee quartiles and prevalent metabolic syndrome . with the lowest quartile of paee as reference , there was a strong inverse association between paee and prevalent metabolic syndrome . compared with the lowest quartile of paee , being in the 2nd quartile of paee was associated with a reduction in the risk of prevalent metabolic syndrome , which was not statistically significant ( odds ratio 0.63 [ 95% ci 0.321.25 ] ) . the risk of prevalent metabolic syndrome was significantly lower for the 3rd and 4th quartiles of paee compared with the 1st quartile ( 0.32 [ 0.14 0.73 ] and 0.19 [ 0.08 0.49 ] , respectively ) . multivariate adjustments for age , sex , residential area , smoking , alcohol drinking , fruit and vegetable consumption , and educational level as potential confounders attenuated but did not change these associations . independent associations between paee and metabolic syndrome in adult cameroonians ( n = 552 ) data are odds ratio or odds ratio ( 95% ci ) . adjusted for age , sex , residential area , smoking , alcohol consumption , fruit and vegetable consumption , and number of years of education . defining the lowest quartile of paee to represent sedentary or inactive lifestyle , the population attributable fraction of prevalent metabolic syndrome due to a sedentary or inactive lifestyle was 26.3% ( 25.3% in women and 35.7% in men ) . expressing paee as a continuous variable , and assuming a linear relationship with the metabolic syndrome , each kj / kg / day of paee was associated with a 2.1% lower risk of prevalent metabolic syndrome ( odds ratio 0.98 , p = 0.03 ) , after adjustment for potential confounders as in table 2 , model 3 . this implies that an increase of 6.5 kj / kg / day of paee would correspond to approximately 13.7% risk reduction in prevalent metabolic syndrome . this amount of paee can be achieved by 30 min / day of brisk walking . our results suggest that free - living paee was significantly and independently associated with prevalent metabolic syndrome . this association persisted after multivariate adjustments for possible confounders and was similar in both urban and rural dwellers , even though rural dwellers were more physically active and had a more favorable metabolic profile than urban dwellers . low levels of physical activity may be contributing significantly to the metabolic syndrome in this population . theoretically , these data suggest a 26.3% population attributable fraction of prevalent metabolic syndrome as a result of being in the lowest quartile of paee . however , independent of several confounders , it is likely that small changes in paee may confer significant benefits in terms of lower risk of metabolic syndrome . because of the cross - sectional design of our study , we can not infer causality . the study sample included only adults aged 2555 years without known metabolic syndrome or cvd . therefore , these results may not be directly generalizable to younger or older individuals , as well as individuals with diagnosed prevalent metabolic or cardiovascular disease . the lack of complete population registers as well as the absence of fixed house addresses or telephone numbers limited our ability to use simple random sampling of participants . our use of volunteers from a study - established sampling frame may introduce sampling bias , which would limit the generalizability of these results even to our target population . however , our sample was similar to other larger population - based surveys with respect to bmi , blood pressure , and educational level ( 21,22 ) . separate from prevalence of risk factors , one could argue that educational level may have influenced volunteering for this study . however , the proportion of volunteers in this sample having at least a secondary education ( 44% of men and 35% of women ) was comparable to data from the 2004 demographic and health survey in cameroon ( 48% of men and 32% of women ) ( 21 ) . the observed inverse association between total volume of physical activity ( paee ) with the metabolic syndrome is comparable to results from previous studies which used objective measures of physical activity ( 12,13,23 ) ( 23 ) reported a significant inverse association between paee scaled for fat - free body mass and a clustered metabolic syndrome score . however , we scaled paee for body weight , not fat - free mass . although researchers agree that paee should be expressed taking into account differences in body size , the exact scaling factor applicable to body size is still a contentious issue . during light intensity locomotion activities , which are the main component of total activity in many people , the expression of paee per kilogram body weight may be more appropriate for the habitual free - living activity pattern , and is easy to interpret for clinical or public health usage . the importance of the distribution of free - living physical activity intensities , beyond total volume of physical activity , on metabolic health is still uncertain . many studies that have examined the effect of intensity distribution of physical activity on global metabolic risk have used self - report of physical activity ( 24 ) . studies that have used objective assessment of physical activity intensity distribution are less common ( 12,13 ) . most studies confirm the deleterious effect of increasing sedentary time or decreasing moderate - to - vigorous physical activity on the metabolic syndrome ( 12,24 ) . however , the benefits of increasing overall activity compared with shifting the intensity distribution toward more vigorous activity is still inconclusive . in this study we show that objectively measured total volume of physical activity had a significant independent association with the metabolic syndrome . our use of total volume of activity may be more applicable to health promotion initiatives in regions with low literacy . it may be more feasible to encourage overall physical activity than to recommend specific subdimensions of activity to less educated people . rapid urbanization of lifestyle in sub - saharan africa is characterized by increasing prevalence and clustering of metabolic risk factors such as diabetes , high blood pressure , and obesity ( 4,25 ) . our data show an evident rural - urban gradient of paee , which mirrors progression toward western levels of habitual physical activity . this transition is not only explained by increasing frequency of high risk individuals , but by a shift in the underlying population distribution of risk factors . our use of an aggregate variable for metabolic health ( the metabolic syndrome ) is relevant for assessing the effects of physical activity level on multiple risk factors in the whole population . further , the metabolic syndrome is associated with a higher morbidity and mortality ( 3 ) . we did not collect data on energy intake in this study because of the difficulties of collecting objective dietary data in a population - based study . however , we adjusted for self - reported fruit and vegetable intake in the analyses , which would capture part of the variance associated with healthy or unhealthy dietary habits . these data suggest a significant population burden of prevalent metabolic syndrome associated with being in the lowest quartile of paee , or a mean paee of 26 7 this level of physical activity corresponds to an average of 45 met - hours / week . typically , these individuals would be spending more than 80% of their time in sedentary activities with very little physical effort . encouraging people to increase their physical activity , even through reduction of sedentary time , may confer considerable health benefits especially in the least active individuals . in conclusion , reduction in physically active lifestyles as a result of urbanization may be critical in driving the current transition toward a higher burden of metabolic diseases in this region . public health interventions to increase physical activity may be expected to significantly curb the burden of metabolic syndrome in sub - saharan africa .

objectiveto evaluate the feasibility of a wearable artificial pancreas system , the diabetes assistant ( dias ) , which uses a smart phone as a closed - loop control platform.research design and methodstwenty patients with type 1 diabetes were enrolled at the universities of padova , montpellier , and virginia and at sansum diabetes research institute . each trial continued for 42 h. the united states studies were conducted entirely in outpatient setting ( e.g. , hotel or guest house ) ; studies in italy and france were hybrid hospital hotel admissions . a continuous glucose monitoring / pump system ( dexcom seven plus / omnipod ) was placed on the subject and was connected to dias . the patient operated the system via the dias user interface in open - loop mode ( first 14 h of study ) , switching to closed - loop for the remaining 28 h. study personnel monitored remotely via 3 g or wifi connection to dias and were available on site for assistance.resultsthe total duration of proper system communication functioning was 807.5 h ( 274 h in open - loop and 533.5 h in closed - loop ) , which represented 97.7% of the total possible time from admission to discharge . this exceeded the predetermined primary end point of 80% system functionality.conclusionsthis study demonstrated that a contemporary smart phone is capable of running outpatient closed - loop control and introduced a prototype system ( dias ) for further investigation . following this proof of concept , future steps should include equipping insulin pumps and sensors with wireless capabilities , as well as studies focusing on control efficacy and patient - oriented clinical outcomes . objectivewe examined the independent associations between objectively measured free - living physical activity energy expenditure ( paee ) and the metabolic syndrome in adults in rural and urban cameroon.research design and methodspaee was measured in 552 rural and urban dwellers using combined heart rate and movement sensing over 7 continuous days . the metabolic syndrome was defined using the national cholesterol education program - adult treatment panel iii criteria.resultsurban dwellers had a significantly lower paee than rural dwellers ( 44.2 21.0 vs. 59.6 23.7 kj / kg / day , p < 0.001 ) and a higher prevalence of the metabolic syndrome ( 17.7 vs. 3.5% , p < 0.001 ) . in multivariate regression models adjusted for possible confounders , each kj / kg / day of paee was associated with a 2.1% lower risk of prevalent metabolic syndrome ( odds ratio 0.98 , p = 0.03 ) . this implies a 6.5 kj / kg / day difference in paee , equivalent to 30 min / day of brisk walking , corresponds to a 13.7% lower risk of prevalent metabolic syndrome . the population attributable fraction of prevalent metabolic syndrome due to being in the lowest quartile of paee was 26.3% ( 25.3% in women and 35.7% in men).conclusionsurban compared with rural residence is associated with lower paee and higher prevalence of metabolic syndrome . paee is strongly independently associated with prevalent metabolic syndrome in adult cameroonians . modest population - wide changes in paee may have significant benefits in terms of reducing the emerging burden of metabolic diseases in sub - saharan africa .

cardiovascular disease ( cvd ) and diabetic nephropathy ( dn ) are the major causes of mortality in individuals with type 1 diabetes ( 13 ) , and a growing body of evidence from basic and clinical studies ( 47 ) supports the relationship between cvd and renal function in health and disease . cvd and dn have been proposed to be manifestations of the same underlying pathology and also exist as interrelated risk factors ( 8,9 ) . the increased mesangial matrix associated with dn is similar to the pathophysiology of coronary atherosclerosis ( 8) . while atherosclerosis tends to remain subclinical until adulthood in individuals with type 1 diabetes ( 10 ) , adolescents with type 1 diabetes demonstrate reduced peak exercise capacity and decreased cardiac function compared with nondiabetic adolescents ( 11 ) . although the mechanisms underlying the reduced peak exercise capacity are poorly understood , we have previously shown that insulin resistance and elevated ldl cholesterol ( ldl - c ) were associated with low peak volume of oxygen ( vo2peak ) ( 11 ) . elevated albumin - to - creatinine ratio ( acr ) and glomerular filtration rate ( gfr ) , which are early manifestations of dn , are also increasingly recognized in children and adolescents with type 1 diabetes ( 1,12 ) . increased gfr was shown to be associated with increased cardiovascular mortality in adults ( 13 ) . little is known , however , about the possible associations between early renal health and exercise capacity in adolescents with type 1 diabetes , and whether this relationship is independent of insulin sensitivity . accordingly , we sought to examine the associations between markers of renal health and peak exercise capacity in adolescents with type 1 diabetes . we hypothesized that cardiopulmonary fitness would be directly associated with acr and inversely related to estimated gfr ( egfr ) in adolescents with type 1 diabetes . pubertal adolescents between the ages of 12 and 19 years were recruited for a study of diabetes and insulin resistance in youth from type 1 diabetes clinics at the barbara davis center for diabetes with advertisements . control subjects were recruited from advertisements on the university of colorado anschutz medical campus , and in endocrine , adolescent medicine , and pediatric clinics at children s hospital colorado . sixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects had data available for analyses of cardiopulmonary fitness , insulin sensitivity , acr , and egfr , as determined by creatinine and cystatin c levels . screening included a medical history , physical examination , tanner staging , and fasting laboratory testing ( measurement of glycosylated hemoglobin [ hba1c ] and ldl - c levels ) . type 1 diabetes was defined by american diabetes association criteria plus the presence of glutamic acid decarboxylase and islet cell or insulin autoantibodies , as well as an insulin requirement . the absence of diabetes was confirmed in control subjects by a 2-h , 75-g oral glucose tolerance test . inclusion criteria included tanner stage > 1 and sedentary status ( <3 h regular exercise / week ) to minimize pubertal and training effects . study exclusions included resting blood pressure > 140/90 mmhg or > 190/100 mmhg during exercise , hemoglobin level < 9 mg / dl , serum creatinine level > 1.5 mg / dl , hba1c level > 11% ( 97 mmol / mol ) , smoking , medication - dependent asthma or other conditions precluding exercise testing , use of antihypertensive medications and oral contraceptives , pregnancy , breastfeeding , plans to alter exercise or diet during the study , family history of type 2 diabetes , and use of medications affecting insulin sensitivity ( e.g. , oral or inhaled steroids , metformin , thiazolidinediones , or atypical antipsychotic agents ) . pubertal development was assessed by a single pediatric endocrinologist using the criteria established by tanner and marshall for pubic hair and breast development ( 14 ) . all subsequent tests were performed after a 12-h fast , preceded by 3 days of restricted physical activity and a fixed - macronutrient , weight - maintenance diet ( 55% carbohydrates , 30% fat , 15% protein ) . participants with type 1 diabetes were instructed to monitor blood glucose levels at least four times per day and were excluded if large urine ketones were present on admission to the study . the study was approved by the university of colorado denver institutional review board , and appropriate consent and assent were obtained . a 3-day pediatric physical activity recall questionnaire was used to estimate habitual physical activity ( 11,15 ) , reported as a 3-d average of daily metabolic equivalents . for all bicycle tests , vo2 , volume of carbon dioxide , and minute ventilation were measured , breath - by - breath , at rest and during exercise . arm blood pressures ( by auscultation ) and heart rates ( measured by 12-lead electrocardiogram ) were obtained every minute during exercise . the respiratory exchange ratio was calculated as the volume of carbon dioxide - to - vo2 ratio . subjects were excluded if the peak respiratory exchange ratio was 1.1 . to determine vo2peak in all participants , a graded bicycle riding protocol was performed until subject exhaustion , and was carried out , as previously reported ( 11 ) , on a cycle ergometer ( medgraphics ; medical graphics corp . , st . paul , mn ) while breathing into the mouthpiece of the metabolic cart ( medgraphics cpx / d ; medical graphics corp . ) . vo2peak was reported in milliliters per kilogram per minute and milliliters per lean kilogram determined from dexa scan per minute , as previously reported ( 11,15 ) . blood sugar levels were closely monitored in participants with type 1 diabetes , and short - acting insulin or carbohydrates were administered to achieve a goal pre - exercise range of 100150 mg / dl . insulin sensitivity ( glucose infusion rate [ gir ] in milligrams per kilogram per minute ) was calculated from a hyperinsulinemic - euglycemic clamp ( 80 mu m min insulin ) after an overnight intravenous insulin infusion to normalize glycemia , as previously described ( 11,15 ) . samples for serum creatinine and cystatin c measurements were collected after 34 h of euglycemia ( 100 mg / dl ) and 16 h of bed rest , eliminating the effects of acute glycemia and exercise on egfr ( 1618 ) . the cystatin c and creatinine samples were analyzed in batches , reducing the effects of temporal systemic shifts on the assays . serum cystatin c levels were measured by immunoturbidimetric methodology ( kamiya biomedical ) , and serum creatinine levels were measured by enzymatic methodology ( beckman coulter ) . because of the absence of chronic kidney disease and expected normal to elevated gfrs for age , we used the bouvet equation to estimate gfr : egfr = 63.2 ( serum creatinine/96 ) ( serum cystatin c/1.2 ) ( weight/45 ) ( age/14 ) ( 19 ) . this equation has shown to have the best performance in estimating gfr in adolescents without chronic kidney disease ( 19 ) . spot urine samples were also collected upon study admission for measurement of urinary albumin and creatinine levels , and acr was calculated . analyses were performed in sas ( version 9.3 for windows ; sas institute , cary , nc ) . differences between continuous parametric variables were examined with the t test , continuous nonparametric variables were examined with the wilcoxon signed rank test , and dichotomous variables were examined with the test . egfrs determined by the bouvet equation ( low < 94 , middle range 94106 , and high > 106 ml / min/1.73 m ) and acr anova with a tukey - kramer p value adjustment was used for the comparison of continuous variables across the three groups ( low , middle , and high tertiles ) , and least squares means ( lsms ) were calculated for the tertile groups . pearson correlation , and univariate and multivariable linear regression models were used to examine the associations between egfr determined by bouvet equation and the natural log of acr ( log transformed for these analyses because of skewed distribution ) and vo2peak , unadjusted and adjusted for tanner stage , sex , insulin sensitivity , hba1c level , systolic blood pressure ( sbp ) , and ldl - c level . pubertal adolescents between the ages of 12 and 19 years were recruited for a study of diabetes and insulin resistance in youth from type 1 diabetes clinics at the barbara davis center for diabetes with advertisements . control subjects were recruited from advertisements on the university of colorado anschutz medical campus , and in endocrine , adolescent medicine , and pediatric clinics at children s hospital colorado . sixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects had data available for analyses of cardiopulmonary fitness , insulin sensitivity , acr , and egfr , as determined by creatinine and cystatin c levels . screening included a medical history , physical examination , tanner staging , and fasting laboratory testing ( measurement of glycosylated hemoglobin [ hba1c ] and ldl - c levels ) . type 1 diabetes was defined by american diabetes association criteria plus the presence of glutamic acid decarboxylase and islet cell or insulin autoantibodies , as well as an insulin requirement . the absence of diabetes was confirmed in control subjects by a 2-h , 75-g oral glucose tolerance test . inclusion criteria included tanner stage > 1 and sedentary status ( <3 h regular exercise / week ) to minimize pubertal and training effects . study exclusions included resting blood pressure > 140/90 mmhg or > 190/100 mmhg during exercise , hemoglobin level < 9 mg / dl , serum creatinine level > 1.5 mg / dl , hba1c level > 11% ( 97 mmol / mol ) , smoking , medication - dependent asthma or other conditions precluding exercise testing , use of antihypertensive medications and oral contraceptives , pregnancy , breastfeeding , plans to alter exercise or diet during the study , family history of type 2 diabetes , and use of medications affecting insulin sensitivity ( e.g. , oral or inhaled steroids , metformin , thiazolidinediones , or atypical antipsychotic agents ) . pubertal development was assessed by a single pediatric endocrinologist using the criteria established by tanner and marshall for pubic hair and breast development ( 14 ) . all subsequent tests were performed after a 12-h fast , preceded by 3 days of restricted physical activity and a fixed - macronutrient , weight - maintenance diet ( 55% carbohydrates , 30% fat , 15% protein ) . participants with type 1 diabetes were instructed to monitor blood glucose levels at least four times per day and were excluded if large urine ketones were present on admission to the study . the study was approved by the university of colorado denver institutional review board , and appropriate consent and assent were obtained . a 3-day pediatric physical activity recall questionnaire was used to estimate habitual physical activity ( 11,15 ) , reported as a 3-d average of daily metabolic equivalents . measurements were made during bicycle ergometer testing using a metabolic cart . for all bicycle tests , vo2 , volume of carbon dioxide , and minute ventilation were measured , breath - by - breath , at rest and during exercise . arm blood pressures ( by auscultation ) and heart rates ( measured by 12-lead electrocardiogram ) were obtained every minute during exercise . the respiratory exchange ratio was calculated as the volume of carbon dioxide - to - vo2 ratio . subjects were excluded if the peak respiratory exchange ratio was 1.1 . to determine vo2peak in all participants , a graded bicycle riding protocol was performed until subject exhaustion , and was carried out , as previously reported ( 11 ) , on a cycle ergometer ( medgraphics ; medical graphics corp . , st . paul , mn ) while breathing into the mouthpiece of the metabolic cart ( medgraphics cpx / d ; medical graphics corp . ) . vo2peak was reported in milliliters per kilogram per minute and milliliters per lean kilogram determined from dexa scan per minute , as previously reported ( 11,15 ) . blood sugar levels were closely monitored in participants with type 1 diabetes , and short - acting insulin or carbohydrates were administered to achieve a goal pre - exercise range of 100150 mg / dl . insulin sensitivity ( glucose infusion rate [ gir ] in milligrams per kilogram per minute ) was calculated from a hyperinsulinemic - euglycemic clamp ( 80 mu m min insulin ) after an overnight intravenous insulin infusion to normalize glycemia , as previously described ( 11,15 ) . samples for serum creatinine and cystatin c measurements were collected after 34 h of euglycemia ( 100 mg / dl ) and 16 h of bed rest , eliminating the effects of acute glycemia and exercise on egfr ( 1618 ) . the cystatin c and creatinine samples were analyzed in batches , reducing the effects of temporal systemic shifts on the assays . serum cystatin c levels were measured by immunoturbidimetric methodology ( kamiya biomedical ) , and serum creatinine levels were measured by enzymatic methodology ( beckman coulter ) . because of the absence of chronic kidney disease and expected normal to elevated gfrs for age , we used the bouvet equation to estimate gfr : egfr = 63.2 ( serum creatinine/96 ) ( serum cystatin c/1.2 ) ( weight/45 ) ( age/14 ) ( 19 ) . this equation has shown to have the best performance in estimating gfr in adolescents without chronic kidney disease ( 19 ) . spot urine samples were also collected upon study admission for measurement of urinary albumin and creatinine levels , and acr was calculated . analyses were performed in sas ( version 9.3 for windows ; sas institute , cary , nc ) . differences between continuous parametric variables were examined with the t test , continuous nonparametric variables were examined with the wilcoxon signed rank test , and dichotomous variables were examined with the test . egfrs determined by the bouvet equation ( low < 94 , middle range 94106 , and high > 106 ml / min/1.73 m ) and acr ( low < 5.50 , middle range 5.509.13 , and high > 9.13 mg / g ) were stratified into tertiles . anova with a tukey - kramer p value adjustment was used for the comparison of continuous variables across the three groups ( low , middle , and high tertiles ) , and least squares means ( lsms ) were calculated for the tertile groups . pearson correlation , and univariate and multivariable linear regression models were used to examine the associations between egfr determined by bouvet equation and the natural log of acr ( log transformed for these analyses because of skewed distribution ) and vo2peak , unadjusted and adjusted for tanner stage , sex , insulin sensitivity , hba1c level , systolic blood pressure ( sbp ) , and ldl - c level . adolescents with type 1 diabetes had reduced peak exercise capacity compared with nondiabetic control subjects ( vo2peak 31.5 6.3 vs. 36.2 7.9 ml / kg min , p = 0.046 ; vo2peak / lean kg 43.7 7.0 vs. 51.0 8.6 ml / lean kg min , p = 0.007 ) , despite similar age , tanner stage , bmi , and habitual level of physical activity . when adjusting for egfr , the differences in tanner stage and sex - adjusted means for vo2peak / kg ( lsms se 32.3 1.0 vs. 34.2 1.5 , p = 0.30 ) and vo2peak / lean kg ( lsms se 49.0 2.1 vs. 45.0 1.4 , p = 0.09 ) between adolescents with and without type 1 diabetes lost significance . characteristics for adolescents with type 1 diabetes data are reported as lsms se , unless otherwise indicated . data are reported as the median ( q25q75 ) . in adolescents with type 1 diabetes , egfr correlated strongly with vo2peak ( r = 0.55 , r = 30.22% , p = 0.002 ) and vo2peak / lean kg ( r = 0.44 , r = 19.58% , p = 0.02 ) , and remained significantly associated with vo2peak and vo2peak / lean kg after adjusting for sex and tanner stage ( tables 2 and 3 ) . additional adjustments for insulin sensitivity , hba1c level , sbp , or ldl - c level did not attenuate the significance of the associations among egfr , vo2peak , and vo2peak / lean kg ; however , the association between egfr and vo2peak / lean kg became less significant ( p = 0.048 ; table 3 ) . in contrast , the associations between egfr and vo2peak / kg and vo2peak / lean kg in adolescent control subjects were not significant in fully adjusted models ( data not shown ) . in contrast to egfr , lnacr did not correlate with vo2peak ( se 1.42 1.49 , p = 0.35 ) and vo2peak / lean kg ( se 2.24 1.71 , p = 0.20 ) when adjusted for sex and tanner stage . associations between egfr and vo2peak / kg data are reported as se ; p value . age was not adjusted for because it is part of the bouvet egfr equation . * adjusted for tanner stage , sex , gir , hba1c level , sbp , and ldl - c level . associations between egfr and vo2peak / lean kg data are reported as se ; p value . adjusted for tanner stage , sex , gir , hba1c level , sbp , and ldl - c level . when stratifying egfrs into tertiles , subjects in the highest egfr tertile had significantly lower vo2peak ( lsm se 26.91 1.52 vs. 35.53 1.88 , p = 0.002 ) and vo2peak / lean kg ( lsm se 39.65 2.07 vs. 46.18 2.55 , p = 0.048 ) adjusted for sex and tanner stage , compared with those in the lowest egfr tertile ( fig . 1 ) . stratifying acr into tertiles demonstrated no differences in vo2peak or vo2peak / lean kg among the three groups ( data not shown ) . tanner stage and sex - adjusted means for vo2peak / kg and vo2peak / lean kg stratified by tertiles of egfr . our major observation in this study was that in adolescents with type 1 diabetes , a disease characterized by reduced exercise capacity , egfr was independently and negatively associated with cardiopulmonary fitness , independent of insulin sensitivity and other important risk factors . we previously demonstrated reduced peak exercise capacity , and cardiac and vascular function in otherwise healthy , nonobese adolescents with type 1 diabetes , compared with well - matched nondiabetic control subjects of similar bmi , pubertal stage , and habitual level of physical activity ( 11 ) . furthermore , we found that insulin sensitivity correlated strongly with vo2peak in youths with type 1 diabetes , as did ldl - c level to a lesser degree ( 11 ) . in this report , we present for the first time an independent relationship between renal health and cardiopulmonary fitness that is independent of insulin sensitivity , ldl - c level , and other important covariates . this association may represent a novel pathway of a cardiorenal connection in type 1 diabetes separate from atherosclerosis . type 1 diabetes is associated with shortened life span , and cardiac and vascular dysfunction , independent of coronary artery disease ( 20 ) . in fact , low fitness levels in adults with and without diabetes are associated with cvd mortality and decreased longevity ( 21,22 ) . control of conventional risk factors such as glucose level , cholesterol level , and blood pressure is important , but does not abolish the cardiovascular risk in subjects with type 1 diabetes ( 2,23 ) . although not without controversy , the finnish diabetic nephropathy study ( 24 ) and the pittsburgh epidemiology of diabetes complications study ( 3 ) demonstrated no increase in mortality in adults with type 1 diabetes compared with nondiabetic control subjects in the absence of dn . in contrast , the coronary artery calcification in type 1 diabetes study ( 9 ) demonstrated strong associations among egfr , acr , and progression of coronary artery calcification in adults with type 1 diabetes in the absence of established dn , suggesting that renal health may be associated with early cardiovascular pathophysiology that may take years to manifest clinically . the specific mechanisms underlying the association between renal and cardiovascular health , however , remain unclear , but increasing evidence supports the importance of shared risk factors and pathogenic pathways , including atherosclerosis ( 6,7,11,2528 ) . reduced exercise capacity and its association with renal health highlighted in this article , taken together with the established relationship between fitness and premature cvd mortality , may provide an alternative and novel pathway explaining the premature mortality of subjects with type 1 diabetes ( 21,22 ) . there is currently no generally accepted definition for renal hyperfiltration ( 29 ) , but it is increasingly recognized that elevated gfr is an early hemodynamic abnormality seen in diabetes that is linked with an increased risk of dn ( 1 ) . the meta - analysis from the chronic kidney disease prognosis consortium ( 30,31 ) demonstrated significantly greater all - cause mortality and cardiovascular mortality in individuals with the highest gfr values ( > 105 ml / min/1.73 m ) . similarly , the renal iohexol clearance survey in tromso 6 ( renis - t6 ) recently reported ( 13 ) significantly greater odds of both carotid atherosclerosis and left ventricular hypertrophy in participants in the highest gfr quartile ( > 101.2 ml / min/1.73 m ) . it is , however , also plausible that the mechanisms underlying the association between renal health and exercise capacity in adolescents with type 1 diabetes differ from the link seen with atherosclerosis . the mechanisms responsible for the relationship between renal health and exercise capacity are not yet clear . adults with type 1 diabetes and elevated gfr demonstrate cardiovascular dysfunction , including increases in arterial stiffness and altered flow - mediated dilatation ( 32,33 ) . we previously demonstrated reduced forearm blood flow in youths with type 1 diabetes ( 11 ) as evidence of vascular dysfunction , which correlated with reduced vo2peak . moreover , alterations in arterial stiffness have also been reported to be present in youths with type 1 diabetes ( 34 ) . elevated gfr is associated with endothelial dysfunction via cyclooxygenase-2 activity and nitric oxide synthase inhibition , and relatively higher blood pressures ( 3537 ) , both of which could directly impact exercise capacity . elevated gfr is also associated with increased carotid intimal medial thickness and left ventricular hypertrophy in nondiabetic individuals , further suggesting that increased gfr potentially reflects a generalized change in vascular function rather than simply reflecting intrarenal abnormalities ( 13 ) . endothelial dysfunction could also contribute to elevated gfr , thus the relationships could be bidirectional . we did not observe a significant association between acr and peak exercise capacity , potentially because increasing evidence supports the distinction of albuminuria as a risk factor for dn and not an early phenotype of dn ( 38 ) . furthermore , the prevalence of microalbuminuria ( n = 9 ) was low due to the young age of our population . . however , to minimize the effect of sample size , we used careful and detailed physiological measurements , including gold standard fitness testing and hyperinsulinemic - euglycemic clamp studies . we also chose groups similar in habitual physical activity , pubertal stage , sex , and bmi , and controlled for prestudy diet and physical activity . another limitation to the current study included the cross - sectional design , which prevents the determination of causality , and whether the association holds true longitudinally ; for that reason , the data should be viewed as hypothesis generating . the direction of the association between renal health and exercise capacity ( i.e. , whether renal health leads to reduced exercise capacity or vice versa ) is also difficult to determine with the available data . results from this study may also not be generalizable to older people with type 1 diabetes or to cohorts with a different ethnic distribution . finally , we used an estimate of gfr to assess renal function ; however , to estimate the gfr we used the bouvet equation , which was shown to have the best performance in estimating gfr in children and adolescents with normal to elevated gfrs ( 18 ) . the mean egfr in our lean nondiabetic control subjects is slightly lower than gfrs measured by insulin level in adolescents in some studies ( 39 ) , but is consistent with gfrs estimated by creatinine and cystatin c levels in adolescents in large - scale studies ( 40 ) . in conclusion , in adolescents with type 1 diabetes , a disease characterized by reduced peak exercise capacity compared with nondiabetic adolescents , there was a strong relationship between egfr and cardiopulmonary fitness , independent of directly measured insulin sensitivity and other important risk factors . this observation provides further evidence for the broad interactions observed between renal and cardiovascular function in type 1 diabetes . further research is needed to investigate the longitudinal relationships between renal and cardiopulmonary health in adolescents with type 1 diabetes , and to elucidate the specific mechanisms underlying this relationship as potential therapeutic targets . plain films are likely to remain the best method of imaging gas shadows for many years to come and computed tomography scanning , isotope studies and nuclear magnetic resonance are unlikely to play any major role in the initial investigation of the acute abdomen ( field et al1 ) . shortly after the discovery of the x - ray in 1895 , the first x - rays were studied for medical purposes by wilhelm rontgen . during the decades that followed , x - rays were mainly used for detecting fractures and foreign bodies and gradually for the evaluation of various other diseases , such as acute abdominal pain . approximately 4%10% of emergency department visits are due to acute abdominal pain , making it one of the most encountered complaints.2 the wide variety in presentation of symptoms and the broad spectrum of associated diseases complicates isolation of the cause of abdominal pain , which may vary from life - threatening diseases requiring emergency surgery to mild self - limiting causes.2 an early and accurate diagnosis is essential in decision - making , and insufficient workup results in unnecessary interventions or delayed treatment.24 a study performed at the university of virginia in the us compared data from patients with acute abdominal pain at three different time points over a period of 35 years , ie , 1972 , 1992 , and 2007.5 the proportion of patients presenting with acute abdominal pain as a chief complaint remained more or less stable over the years , being 4% in 1972 , 5% in 1992 , and 6.6% in 2007 . plain abdominal radiography was the only diagnostic imaging modality available in 1972 and was ordered in 43% of all patients . in 1992 , ultrasound and computed tomography ( ct ) had come into use but were barely ordered , ie , in only 6.8% of all patients . in 2007 , the use of ultrasound and ct was widespread and the use of plain abdominal radiography decreased somewhat , but was still performed in a considerable proportion of patients ( 21% ) . ct and ultrasound were used liberally , and either one of these tests was performed in 42% of all patients . these data show increased use of ct and ultrasound and decreased use of plain abdominal radiographs ( a decrease of approximately one third ) between 1992 and 2007 . in the same time period , the average time patients spent at the emergency department had risen from 2.9 hours in 1992 to 4.26 hours in 2007 . patients who had undergone a ct scan spent an average of 6.64 hours in the emergency department compared with 3.44 hours without ct evaluation . diagnostic accuracy improved over the years due to increased use of ct and ultrasound ; in 1992 , 41.3% of all patients were diagnosed with nonspecific abdominal pain compared with 21.1% in 2007 . the number of patients admitted to the ward decreased from 27.4% in 1972 to 18.4% in 1992 . factors contributing to improved diagnostic accuracy include establishment of emergency medicine faculties and increased possibilities for laboratory testing . the widespread availability of various diagnostic modalities , such as ct , ultrasound , and nuclear imaging , probably account for the most significant change.57 diagnosing the underlying cause of acute abdominal pain remains a challenge despite the increase in diagnostic accuracy over the years . the ideal diagnostic imaging modality for evaluation of acute abdominal pain in adult patients at the emergency department should provide a balance between the highest diagnostic value and most accurate management changes on one hand , and lowest radiation exposure , discomfort , and duration of stay at the emergency department on the other hand , while ultimately resulting in the lowest cost to the health care system . several studies have demonstrated that a diagnosis based solely on a patient s medical history , physical examination , and laboratory tests is not reliable enough , despite the fact that these aspects are essential parts of the workup of a patient presenting with acute abdominal pain.2,8 further diagnostic workup such as imaging is therefore mandatory in patients suspected of an urgent medical condition . imaging workup traditionally starts with abdominal radiography.9 standard abdominal radiography consists of three views , ie , a supine abdominal view combined with an erect chest film and an upright abdominal view.10,11 uk and us guidelines advise consideration of abdominal radiography in case of hospital admission and or surgery in patients presenting with acute abdominal pain.12,13 one study compared the initial diagnosis after clinical evaluation and plain radiography with the final diagnosis , and found that these diagnoses corresponded in only 502 of a total of 1021 patients ( 49%).3 diagnosis based only on plain abdominal radiography corresponded with the final diagnosis in 514 of the 1021 patients ( 50% ) . the improvement in accuracy of plain abdominal radiography combined with clinical examination was not significant ( p = 0.14 ) . in 117 of the 1021 patients ( 11% ) , the treating physician changed the initial clinical diagnosis after plain abdominal radiography , and these changes were accurate in only 39 cases ( 22% ) . additionally , the level of confidence of the assigned diagnosis was recorded in 983 patients before and after plain abdominal radiography . for 875 patients in whom the diagnosis had not changed , the level of confidence also remained unchanged . several studies have demonstrated a high percentage of plain abdominal radiographs without abnormal or specific findings . two studies demonstrated that 77% and 78% of all requested plain abdominal radiographs showed no abnormal findings.14,15 another study demonstrated that in only 83 of 871 patients ( 10% ) , a specific diagnostic abnormality was noted on plain abdominal radiography.16 a study analyzing the value of plain abdominal radiography in addition to clinical examination showed that management changed in only 15 patients ( 8.9%).17 in 90 of 153 remaining patients ( 53.6% ) , the initial diagnosis changed due to other imaging modalities , and in 63 patients the diagnosis remained unchanged after plain abdominal radiography . whether plain abdominal radiography contributes to therapeutic decision - making or disposition remains questionable . particularly in the case of a negative result , the additional value of plain abdominal radiographs is disputed . it is for this reason that several studies suggest ordering plain abdominal radiographs for specific indications only , in order to reduce the number of unnecessary requests.1113,18,19 specific indications for ordering plain radiography include suspicion of perforated viscus , urinary tract stones , bowel obstruction , and ingested foreign body . the identification of a small amount of free intra - abdominal gas remains one of the most significant signs in medicine . the combination of abdominal pain and a pneumoperitoneum , even in the absence of other clinical signs , will usually lead to a laparotomy in search of a perforated viscus advocates of conventional radiography state that plain abdominal radiography should be the first diagnostic modality used in suspicion of a perforated viscus . it is possible , using careful radiographic technique , to demonstrate as little as 1 ml of free gas on an erect chest or left lateral decubitus abdominal film.20 the high percentage of missed cases is due to technical imperfections rather than limitations of the test ( poor quality of plain abdominal radiography , excluding the uppermost portion of the peritoneal cavity of the image).21 in that study , the radiographs demonstrated pneumoperitoneum in only 51% of patients with documented visceral perforation . left lateral decubitus radiographs demonstrated pneumoperitoneum in 96% of patients , chest radiographs in 85% , and upright and supine abdominal radiographs in 60% and 56% , respectively.22 another study described pneumoperitoneum in only 83% of all patients with documented visceral perforation.23 one study compared the diagnosis of patients suspected of perforated viscus before and after plain abdominal radiography.3 the positive predictive value was not influenced by plain abdominal radiography . the level of confidence in the diagnosis changed in six of 13 ( 46% ) patients with the clinical diagnosis of a perforated viscus . these data were obtained from a cohort of 1021 patients who presented at the emergency department with acute abdominal pain.2 this study demonstrates that plain abdominal radiographs have no added value in the diagnostic workup . several studies have demonstrated that plain abdominal radiography has a lower accuracy than other diagnostic modalities . one study compared ultrasonography with plain abdominal radiography in detection of pneumoperitoneum , and included 188 patients suspected of visceral perforation.24 all patients underwent chest and/or abdominal radiography and ultrasonography in order to detect free intraperitoneal air ; 165 patients had suspicion of pneumoperitoneum after ultrasonography , and in 157 of the suspected patients , visceral perforation was confirmed intraoperatively . after plain radiography , 126 patients were suspected of pneumoperitoneum which was confirmed intraoperatively in 120 of cases . both diagnostic modalities demonstrated high positive predictive value ( 95% in ultrasound and 94% in radiography ) and similar specificity ( 53% ) . ultrasound did have a higher sensitivity ( 92% versus 78% ) , accuracy ( 88% versus 76% ) , and negative predictive value ( 39% versus 20% ) . ct has proven to be the most accurate diagnostic modality in the evaluation of pneumoperitoneum.25 a small study compared ct with plain radiographic evaluation in 13 patients who underwent diagnostic peritoneal lavage due to abdominal trauma.25 only five of 13 patients ( 38% ) demonstrated free air on radiographs compared with 13 of 13 patients on ct . another study retrospectively reviewed ct and plain radiographs ( when available ) of 76 patients with proven perforation of the gastrointestinal tract.26 in 65 of 76 patients , ct was truly positive and in 11 patients was falsely negative . the cause of perforation was correctly predicted in 51 ( 78% ) of all patients , and the location of the perforation was correctly predicted in 55 patients ( 84.6% ) on ct . in 63 patients plain radiographs were used , of which 32 ( 52% ) were truly positive and 31 ( 48% ) were falsely negative for the presence of a perforation . the available evidence demonstrates that even though the positive predictive value of plain abdominal radiography is similar to that of other diagnostic modalities , the sensitivity and negative predictive value are far too low . the additional value of ct lies in the possibility of providing more information on the location and underlying cause of the perforation , or in providing an alternative diagnosis . in current practice , the high number of missed cases of perforation after plain abdominal radiography is unacceptable and renders the additional value of negative plain abdominal radiography very limited . moreover , a plain abdominal radiograph that is positive for free air conveys limited information on the location and underlying cause ; an additional ct scan will often need to be made to be able to adapt the operative strategy to the specific case . performing surgery without adequate information about the perforation site is a conceptual mistake and nowadays should be considered as substandard clinical practice ( not all free air is caused by a perforated gastric or duodenal ulcer ; consider , amongst others , colonic perforation , surgery - related perforation , perforated diverticular disease , perforated appendicitis , inflammatory bowel disease , or endoscopy ) . no place exists for abdominal radiography in the evaluation of patients suspected of visceral perforation or , for that matter , in any patient with acute abdominal pain . the standard imaging modality for detecting urinary tract stones should ideally provide information about size , site , and composition of the ureteral stone and presence of ureteral obstruction . most ureteral stones can be identified as a calcification causing a filling defect or ureteral obstruction on plain abdominal radiography.1 a plain film of the abdomen , including kidney , ureter , and bladder , has shown sensitivity ranging from 44% up to 77% and specificity in detection of stones from 80% to 87%.2729 in one study , the diagnosis of patients suspected of ureteral stones before and after plain abdominal radiography compared with the final diagnosis revealed a correct change in diagnosis in six of 11 patients ( 55%).3 the level of confidence in the diagnosis remained the same as well as having a positive predicting value , which reached 57% after clinical evaluation and 58% after radiographs . another study concluded that plain abdominal radiography had a sensitivity of 45% and specificity of 77% for detection of ureteral calculi.30 in patients previously diagnosed with urolithiasis presenting with similar symptoms , abdominal radiography could be useful to diagnose kidney stones . the advantage of ct over plain abdominal radiography is that an alternative diagnosis may be presented if the suspicion of urolithiasis is not confirmed . additional information concerning urolithiasis may also be obtained by ct , such as size and location , which are both crucial factors in guiding therapy . ct has replaced the use of plain abdominal radiography and intravenous urography in the detection of ureteral stones . bowel obstruction is a common cause of acute abdominal pain . about 7% of all patients with acute abdominal pain imaging in patients suspected of bowel obstruction should provide information about the site , cause , and level of the obstruction.31 plain abdominal radiography is advocated as a standard diagnostic modality for the detection of bowel obstruction.12,13 plain abdominal radiographic findings have been shown to be diagnostic in 50%60% , indifferent in 20%30% , and misleading in 10%20% of patients.32 in one study , the sensitivity of plain abdominal radiography after clinical evaluation was significantly higher than that of clinical evaluation alone ( 74% versus 57% , respectively).3 changes in diagnosis after plain abdominal radiography were correct in only 16 of 24 patients ( 66% ) and the level of confidence remained unchanged in 32 of 71 patients ( 52% ) . the sensitivity of a clinical evaluation combined with a score card for clinical signs / symptoms proved to be similar to a clinical evaluation combined with plain abdominal radiography.3 frager et al compared diagnoses after clinical evaluation combined with plain abdominal radiography or ct.33 in patients with a complete obstruction , ct demonstrated a sensitivity of 100% compared with 46% after plain abdominal radiography . for partial obstruction , ct had a sensitivity of 100% compared with 30% for plain abdominal radiography . of the 61 patients who underwent surgery , 52 patients were confirmed to be correctly diagnosed preoperatively ( 85% ) based on ct findings . the exact location of the obstruction was correctly diagnosed in 50 of 53 patients ( 94% ) on ct . in addition to its higher sensitivity , an important advantage of ct is the ability to provide information about the underlying cause of obstruction or to provide information about an alternative diagnosis if no signs of bowel obstruction are present . additional diagnostic investigations should be considered if clinically relevant , particularly if surgery is considered . most foreign bodies pass through the gastrointestinal tract harmlessly . if patients are symptomatic or if the ingested foreign body is potentially dangerous , additional diagnostic examinations should be undertaken . plain abdominal radiography demonstrates a sensitivity of 90% , specificity of 100% , and accuracy of 100% for ingested foreign bodies , but the foreign body has to be radio - opaque to be seen on plain abdominal radiography.16 there is no evidence available as to whether ct has a higher sensitivity and specificity than plain abdominal radiography in the case of ingested foreign bodies . the advantage of ct is the ability to provide information about the location of the foreign body , which is a prerequisite when surgical treatment is planned . body packers orally ingest , or rectally or vaginally insert packed drugs in order to smuggle them . plain abdominal radiography is used to establish the diagnosis of drug packing and is considered the gold standard.34 if plain abdominal radiography is negative or inconclusive but a high suspicion of body packing remains , a ct scan should be done . the sensitivity of plain abdominal radiography is 85%100% , but ct has a higher sensitivity and additionally provides more accurate information about the number and location of packages . the use of plain abdominal radiography gives rise to a high number of false negative results , which could be due to overprojection of feces or a specific packaging method.35 there is no solid evidence that ct has a higher sensitivity or diagnostic accuracy than plain abdominal radiography . the low negative predictive value of plain abdominal radiography leads to the conclusion that , if clinically relevant , a ct should be used as the diagnostic modality of choice . in spite of the recent proliferation of other imaging techniques , plain films still retain their position as one of the most useful initial investigations imaging techniques such as ct and ultrasound have been shown to increase diagnostic accuracy substantially,5 and consequently have significantly decreased the added diagnostic value of plain abdominal radiography in a clinical setting . despite recent abundant evidence of its limited value , many physicians still rely on plain abdominal radiography as a simple , cheap , and widely available first diagnostic modality with lower radiation exposure than ct . proponents of plain abdominal radiography advocate its use to prevent high radiation exposure in patients due to unnecessary ct imaging . the average plain abdominal radiograph exposes the patient to 0.7 msv and an abdominal ct exposes the patient to 10.0 msv.13,36 new techniques , such as automated dose modulation and an iterative reconstruction algorithm , reduce the ct radiation dose . in everyday clinical practice , physicians differentiate , subconsciously or consciously , between urgent and nonurgent conditions in patients . patients with indeterminant symptoms and low or no suspicion of an urgent condition can be discharged home without additional imaging . patients suspected of urgent conditions require timely identification of the underlying cause and treatment within 24 hours to prevent severe complications . a recent study designed to identify the most effective diagnostic strategy for patients with acute abdominal pain has demonstrated that the highest sensitivity for detecting urgent diagnoses is achieved when ultrasonography is performed in all patients and a ct only in the event of inconclusive or negative ultrasonography ( conditional ct strategy).31 using this strategy , ct is only needed in 49% of patients . even though ct exposes patients to a higher radiation dose , it still remains the preferred standard diagnostic modality if ultrasound fails to diagnose the cause of acute abdominal pain correctly . plain radiography demonstrates low sensitivity and accuracy and is therefore generally unhelpful ; because of its low sensitivity and negative predictive value , it can also be misleading in the workup of acute abdominal pain . although the radiation dose is lower than that of ct , the mediocre test characteristics of plain radiography may delay appropriate treatment and are therefore detrimental . numerous studies have demonstrated low sensitivity and accuracy for plain abdominal radiography in the evaluation of acute abdominal pain as well as for various specific diseases , such as perforated viscus , bowel obstruction , ingested foreign body , and ureteral stones . ct provides a better workup than that achieved with plain abdominal radiography alone , and its benefits lie in improving decision - making for management , adapting the surgical strategy , and possibly even avoiding negative laparotomies . when a new test is developed , it can have three possible roles in relation to the existing situation . the existing test can be replaced , the new test could be added on top of the existing test , or the new test could function as a triage test to distinguish between patients who need further examination and those who can be safely observed without additional examination . if we were to look at abdominal radiography as if it was a newly developed test for adult patients with acute abdominal pain in the emergency department , the evidence presented in this manuscript demonstrates that there is no added value nor could abdominal radiography replace ultrasound or ct . if abdominal radiography were to be used as triage , its main purpose would be to rule out disease in patients who genuinely do not have disease , ie , to prevent further investigations . however , for this purpose , the number of false negatives has to be relatively low and sensitivity should be high , which is not the case for abdominal radiographs.37 based on the abundant available evidence , major advances in diagnostic imaging , and changes in the management of certain diseases , we can conclude that there is no place for plain abdominal radiography in the workup of adult patients with acute abdominal pain in current practice .

objectivediabetic nephropathy and cardiovascular disease are strongly related in adults with type 1 diabetes , yet little is known about this relationship in adolescents prior to the onset of detectable clinical disease . we hypothesized that cardiopulmonary fitness would be directly associated with albumin - to - creatinine ratio ( acr ) and inversely related to estimated glomerular filtration rate ( egfr ) in adolescents with type 1 diabetes.research design and methodssixty - nine adolescents with type 1 diabetes and 13 nondiabetic control subjects of similar pubertal stage and bmi had insulin sensitivity ( glucose infusion rate [ gir ] ) , measured by hyperinsulinemic - euglycemic clamp , and lean body mass , measured by dexa . cardiopulmonary fitness was measured by cycle ergometry to obtain peak volume of oxygen ( vo2peak ) , and renal function was measured by egfr using the bouvet equation ( measuring creatinine and cystatin c levels ) and acr.resultsadolescents ( 15.5 2.2 years of age ) with type 1 diabetes ( 6.3 3.8 years diabetes duration ) had reduced vo2peak ( 31.5 6.3 vs. 36.2 7.9 ml / kg min , p = 0.046 ) and vo2peak / lean kg ( 43.7 7.0 vs. 51.0 8.6 ml / lean kg min , p = 0.007 ) compared with nondiabetic control subjects . egfr was inversely associated with vo2peak and vo2peak / lean kg after adjusting for sex , tanner stage , gir , hba1c level , systolic blood pressure , and ldl cholesterol level ( se , vo2peak : 0.19 0.07 , p = 0.02 ; vo2peak / lean kg : 0.19 0.09 , p = 0.048 ) . moreover , participants in the highest tertile for egfr had significantly lower sex- and tanner - adjusted vo2peak and vo2peak / lean kg compared with participants in the lowest tertile.conclusionsadolescents with type 1 diabetes had reduced exercise capacity , which was strongly associated with renal health , independent of insulin sensitivity . future studies should examine the underlying interrelated pathophysiology in order to identify probable targets for treatment to reduce cardiovascular and renal complications . several studies have demonstrated that a diagnosis based solely on a patient s medical history , physical examination , and laboratory tests is not reliable enough , despite the fact that these aspects are essential parts of the workup of a patient presenting with acute abdominal pain . traditionally , imaging workup starts with abdominal radiography . however , numerous studies have demonstrated low sensitivity and accuracy for plain abdominal radiography in the evaluation of acute abdominal pain as well as various specific diseases such as perforated viscus , bowel obstruction , ingested foreign body , and ureteral stones . computed tomography , and in particular computed tomography after negative ultrasonography , provides a better workup than plain abdominal radiography alone . the benefits of computed tomography lie in decision - making for management , planning of a surgical strategy , and possibly even avoidance of negative laparotomies . based on abundant available evidence , major advances in diagnostic imaging , and changes in the management of certain diseases , we can conclude that there is no place for plain abdominal radiography in the workup of adult patients with acute abdominal pain presenting in the emergency department in current practice .

1 . he had a history of bilateral brow suspension surgery 10 years ago at another institution . the patient had a 30 cm 20 cm pelvic mass located in the presacral area with boundaries between both parailiac regions displacing the rectum and bladder . it was completely excised with a pathology report of myelolipoma . in physical examination , he had hypertrichosis , finger clubbing , and radiologically cortical thickening of the bones fig . the patient presented with bilateral blepharoptosis with coarse skin folds the patient had cortical thickening of the bones and clubbing his best - corrected visual acuity was 7/10 in the right eye ( re ) and 5/10 in the left eye ( le ) . horizontal length of the upper lids of re and le was 46 mm and 41 mm , respectively . vertical fissure heights were 6 mm and 4 mm with margin - reflex distances of 0.5 mm and 0.5 mm , respectively . vertical eyelid contour of the re showed an inverse v shape for the re and a smooth curve for the le . the eyelids were floppy and easily everted . to correct these clinical findings , a surgery which combines vertical tarsal shortening accompanied with horizontal whole eyelid wedge resection and brown suspension 10 mg/0.25 ml triamcinolone acetate was injected into each supratarsal space to decrease the thickness of the tarsus . horizontal wedge resection which is not a part of routine ptosis surgery was performed in this operation with the aim of normalizing the obvious horizontal length of the lids . pathological evaluation revealed epidermal hyperplasia , severe inflammatory changes in subepidermal level , hyperplastic sebaceous glands , and collagen tissue derangement fig . reconstruction of each eyelid was achieved by vertical tarsectomy , horizontal full thickness lid resection , and supratarsal steroid injection . ptosis was assessed with silicone rod frontalis suspension , and resection of excessive skin was performed as in blepharoplasty skin biopsy showing thickening of the dermis with increased collagen content and lymphocytic infiltration ( h and e , 10 ) the postoperative course was uneventful and patient satisfaction is good within the 1 year of follow - up fig . it is characterized by skin thickening , clubbing , hyperhidrosis , and periosteal reaction in the long bones . primary hoa is predominantly a male disease that at least 90% of patients are men . although symptoms may be seen in childhood , it manifests mostly during the fifth decade of life . described the largest number of primary hoa series in the literature that the authors reported that family history was positive in all studied patients . the pathogenesis of hoa is unclear and some studies explained the role of several growth factors in the evolution of the disease . although clinical findings are similar , secondary hoa differs from primary hoa with absence of family history . secondary hoa is an acquired form that is associated with usually lung disease but also heart , liver , and intestines . the presented patient had a huge pelvic mass which was completely excised with a pathology report of myelolipoma . hence , our diagnosis was secondary hoa due to the paraneoplastic manifestations of pelvic malignancy which causes secretion of many growth factors . blepharoptosis may develop secondary to sebaceous gland hyperplasia , thickening of the dermis with increased collagen content , mucin deposition , and lymphocytic infiltration or due to additionally marked scarring . our patient was a 52-years - old man with negative family history which let us exclude the diagnosis of primary hoa . the differential diagnosis includes several diseases as acromegaly , syphilitic periostitis , and thyroid acropachy . acromegaly is characterized by enlargement of facial bones that are not present in our case . serology , radiology , and clinical findings did not support a diagnosis of syphilis or thyroid disease . hoa may develop secondary to pulmonary or congenital cyanotic cardiac diseases those were not present in our patient . the remaining possible etiological factor for secondary hoa in our case was the huge pelvic malignancy which was excised previously . the surgery for these kinds of pathologies may be planned as single or staged procedures . blepharoplasty with excessive skin excision is usually required both for better cosmesis and to reduce the tissue bulk . no complications were encountered during surgery , but bleeding was more than any other lid surgery . the profound inflammatory reaction in the tissue might be the cause of this excessive bleeding . hoa may not be always primary , particularly in patients with negative family history . in cases of findings with abnormal fibroproliferative and inflammatory changes , detailed systemic examination and investigations both using serologic and imaging modalities the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . cyclic organic structures comprise a wide variety of interesting examples in strained , distorted , bent , and sterically hindered molecular systems in nanoscience . these structures have fascinated chemists for several decades not only because of their unique chemical bonding but also because of the potential to tailor their unique physical properties through variations in molecular size , shape , and crystallinity . cyclic nanostructures composed entirely of conjugated carbon subunits are especially fascinating because they exhibit a myriad of interesting electronic properties . in particular , because cycloparaphenylene nanostructures are composed of aromatic subunits with radially oriented p orbitals , these materials can support mobile charge carriers on the delocalized valence and conduction orbitals along the backbone chain . as a result , cycloparaphenylenes and other well - ordered macrocyles may find applications as conducting materials in molecular electronics , organic field - effect transistors ( ofets ) , and nonlinear optics ( nlos ) due to their unique optoelectronic properties . in general , the ability to control and understand the electronic response of organic nanostructures at the molecular level would have a tremendous impact in nanoscale optoelectronic technologies . carbon nanorings large enough to encapsulate fullerenes were first synthesized by kawase et al . and are composed of a conjugated carbon skeleton in a smooth belt - shaped structure . many of the nanorings synthesized by the kawase group are actually part of a larger family of molecules known as phenylacetylene macrocycles ( pams ) , which are composed of alternating phenyl and ethynyl subunits sequentially bonded to form a single ring . the phenyl subunits in pams are usually linked in the meta or para positions and can be composed of four to nine phenyleneethynylene units . in a very recent communication , jasti et al . synthesized and characterized a set of - , - , and -cycloparaphenylenes composed solely of phenyl rings sequentially connected to form a single nanohoop ( figure 1 ) . their synthesis was especially noteworthy because cycloparaphenylenes form the fundamental annular segments of armchair carbon nanotubes and may serve as templates toward the rational synthesis of carbon nanotubes with specific chiralities . [ n]-cycloparaphenylenes with n = 9 , 12 , and 18 . each cycloparaphenylene is composed solely of phenyl rings sequentially connected in the para position to form a single nanoring . a peculiar feature of these cycloparaphenylenes is their unusual optoelectronic properties as a function of molecular size . specifically , as the carbon nanoring size is increased , the lowest computed absorption energy becomes larger . this result is surprising and counterintuitive to our usual expectations ! for example , our chemical intuition from organic chemistry tells us that , as the number of repeat units in a macromolecule increases , the distance between molecular energy levels diminishes , effectively decreasing the optical absorption gap . this trend ( which actually arises from quantum confinement effects ) qualitatively describes the variation of absorbance properties in -conjugated systems as the number of monomer units increase in a polymer . however , in the case of cyclic nanorings , the excitation energies as a function of size seem to contradict this quantum - confinement - effect argument . to rationalize these trends , with a view of understanding the nature of electronic excitations in these nanorings , i carried out ab initio calculations on both cyclic and linear paraphenylenes ( figure 2 ) composed of 5 to 18 phenyl repeat units each . results obtained from time - dependent density functional theory ( tddft ) give electronic band gaps , optical absorption energies , and oscillator strengths that can be used to estimate exciton binding energies as a function of paraphenylene size . the tddft calculations also provide a two - dimensional real - space analysis of transition densities that represent coherent electronic transitions between ground and electronically excited states . these transition densities give a panoramic view of electronhole coherence and exciton delocalization in each of the paraphenylene systems . on the basis of overall trends in exciton binding energies and their spatial delocalization , i find that excitonic effects play a significant role in understanding the unique photoinduced dynamics of cycloparaphenylene nanoring systems . molecular structures and atom labels for ( a ) cyclic paraphenylenes and ( b ) linear paraphenylenes . the specific atom numbers depicted in these figures define an ordered basis for generating the density matrices discussed in section . the cyclic and linear paraphenylenes ( n = 518 ) analyzed in this work are shown in figure 2 . all quantum chemical calculations were carried out with the parameter - free pbe0 hybrid density functional that incorporates a fixed combination of 25% hartreefock exchange and perdew s gga corrections in the correlation contribution . have shown that the use of pure local and gradient - corrected functionals ( i.e. , lda and pbe ) results in unphysical unbound exciton states , whereas hybrid functionals partially overcome this problem by mixing in a fraction of nonlocal hartreefock exchange . on the basis of their studies , i have chosen the pbe0 functional for this work because the pbe0 kernel provides a balanced description of neutral excitons ( both singlets and triplets ) in conjugated polymers and carbon nanotubes . ground - state geometries of all cyclic and linear paraphenylenes were optimized at the pbe0/6 - 31g(d , p ) level of theory . geometry optimizations were calculated without symmetry constraints , and root - mean - squared forces converged to within 0.00003 au . at the optimized ground - state geometries , tddft calculations were performed with a larger , diffuse 6 - 31+g(d , p ) basis set to obtain the lowest four singlet vertical excitations . for both the ground - state and the tddft calculations , i used a high - accuracy lebedev grid consisting of 96 radial and 302 angular points . all ab initio calculations were performed with a locally modified version of gamess . within the tddft formalism , one obtains the excited - state electron density , (r ) |(r)| = i=1n|i(r)| composed of n - occupied molecular orbitals i(r ) as solutions from the time - dependent kohnsham equations . in the same way that one can calculate transition density matrices from time - dependent hartreefock orbitals , one can use orbitals from the noninteracting kohnsham system to form transition densities that include many - body correlation effects from the tddft formalism . , one can construct coordinate qv and momentum pv matrices with elements given by 12 where g and v are ground and excited states , respectively . the fermi operators ci and ci represent the creation and annihilation of an electron in the ith basis set orbital in . for the cyclic and linear paraphenylenes analyzed in this work , the qv and pv matrices each form a two - dimensional xy grid running over all the carbon sites along the x and y axes . the specific ordering of the carbon sites used in this work is shown in figure 2 . the ( pv)mn momentum matrix represents the probability amplitude of an electronhole pair oscillation between carbon sites m and n , and the ( qv)mn coordinate matrix gives a measure of exciton delocalization between sites m and n. each of these matrices provides a global view of electronhole coherence and exciton delocalization for optical transitions within the paraphenylene systems . in the ground - state optimizations for the linear paraphenylenes , the dihedral angle between adjacent benzene rings was calculated to be 37. this is close to the dihedral angle in isolated biphenyl and is consistent in all of the linear paraphenylenes regardless of length . in contrast , for the smallest and highly strained n = 5 cyclic paraphenylene , the benzene rings adopt a wide variety of smaller dihedral angles between 9 and 27. figure 3 shows the average dihedral angle between adjacent benzene rings as a function of paraphenylene size . for a given number of benzene rings , the average dihedral angle in the cyclic paraphenylenes is always smaller than their acyclic counterparts . however , as the size of the cyclic paraphenylene increases , the strain energy becomes smaller , and the dihedral angles between benzene rings increase to 36. the optimized cartesian coordinates for both the cyclic and the linear paraphenylenes can be found in the supporting information . average dihedral angle between adjacent benzene rings as a function of paraphenylene size . as the number of benzene rings increases , the average dihedral angle for the cyclic paraphenylenes asymptotically approaches the acyclic dihedral angle of 37. to investigate optoelectronic trends as a function of size and shape , optical absorption energies , eopt , were computed for all the cyclic and linear ground - state geometries . table 1 compares the lowest excitation energies and oscillator strengths between the cyclic and linear paraphenylenes , and figure 4 displays eopt as a function of size . the other higher - lying singlet excitations ( up to s4 ) can be found in the supporting information . as the number of benzene rings increases , the lowest excitation energies for both the cyclic and the linear paraphenylenes asymptotically approach a value of 3.48 ev . however , as figure 4 illustrates , the manner in which they approach this asymptotic value is considerably different : eopt for the cyclic systems increases with size , whereas eopt for the acyclic systems decreases . optical excitation energies ( eopt ) for the cyclic and acyclic paraphenylenes as a function of the number of benzene rings . eopt for the cyclic systems increases with size , whereas eopt for the acyclic systems decreases . all excitation energies were obtained from pbe0/6 - 31+g(d , p ) tddft calculations at pbe0/6 - 31g(d , p)-optimized geometries . another significant difference between the two systems is the variation of oscillator strengths as a function of size . for the linear paraphenylenes , the oscillator strengths increase linearly as a function of chain length , whereas the oscillator strengths for most of the even - membered cyclic systems are zero ( due to molecular symmetry ) . although the dft geometry optimizations were performed without symmetry constraints , most of the calculations for the smaller ( n 12 ) even - membered nanorings converged to a c2v - like symmetric structure . for nanorings containing an odd number of benzenes , the optimized structures have a reduced symmetry , and the oscillator strengths are nonzero . however , as the number of benzene rings increases , the strain energy becomes smaller , and several conformers with different dihedral angles can exist with similar energies . i found that these different conformers have various oscillator strengths ( due to reduced symmetry ) but nearly indentical energies . a complete analysis of the conformational landscape available to the nanorings is beyond the scope of this work and would have a negligible effect on the excitation energies in the larger systems . the small s1 s0 oscillator strengths obtained for the nanorings also have a close analogy with the very recent study by kilina et al . that calculated optoelectronic properties of finite - length carbon nanotubes . in their study , it was found that hybrid functionals determine the lowest singlet - excited state to be an optically inactive ( dark ) state with the optically active ( bright ) state lying higher in energy . similarly , for the large ( n 12 ) nanorings in this work , the second and third singlet excitations are bright states with strongly allowed transitions ( table si-1 in the supporting information ) . these trends in oscillator strengths for the nanorings can also be explained qualitatively in terms of a simple exciton model where one transition dipole moment is assigned to each benzene ring ( figure 5 ) . for the lowest s1 s0 excitation , the transition moments in both the linear and the cyclic paraphenylenes are aligned in a head - to - tail arrangement . however , because of their circular geometries , the s1 s0 transition moments in the cyclic paraphenylenes effectively cancel , while the total transition moment increases as a function of length in the linear systems ( for the larger , more flexible nanorings , some of the transition dipole moments have a small component perpendicular to the ring , and the vectorial sum is slightly nonzero ) . in contrast , for the other higher - lying singlet excitations , the transition dipoles are aligned in one direction for half of the nanoring and in the opposite direction for the other half of the ring , producing a net transition dipole and a nonzero oscillator strength . as a result , the structural difference between cyclic and linear geometries imposes additional symmetry constraints that determine the oscillator strengths in these conjugated systems . arrangements of individual transition dipole moments for ( a ) cyclic and ( b ) linear paraphenylenes during the s1 s0 excitation . the transition moments in the cyclic geometry effectively cancel , but the total transition moment increases proportionally with length in the linear systems . to provide further insight into these optoelectronic trends , i carried out a two - dimensional real - space analysis of density matrices for both the cyclic and the linear systems . figure 6 displays the absolute values of the coordinate density matrix elements , |(q1)mn| , for the lowest excitation energy ( s1 s0 ) in the n = 5 , 9 , 14 , and 18 paraphenylenes . the coordinate and momentum , |(p1)mn| , density matrices for all of the other paraphenylenes the x and y axes represent the benzene repeat units in the molecule , and the individual matrix elements are depicted by the various colors . contour plots of coordinate density matrices ( q1 ) for the lowest excitation energy ( s1 s0 ) in the cyclic and linear paraphenylenes . the x- and y - axis labels represent the number of benzene repeat units in the molecule . the elements of the coordinate matrix , qmn , give a measure of exciton delocalization between sites m ( x axis ) and n ( y axis ) . although the cyclic and linear paraphenylenes are composed of similar benzene repeat units , the density - matrix delocalization patterns in each system are considerably different . for the linear systems , the electronhole pair created upon optical excitation becomes primarily localized in the middle of the molecule and away from the edges . in contrast , the density matrices for the cyclic systems have significant off - diagonal elements that persist even for the largest n = 18 nanoring . the magnitude of the off - diagonal elements represents electronic coherence between different atoms , and figure 6 shows that the electronhole states are delocalized over the entire circumference in the cyclic systems . the coherence size , which characterizes the distance between an electron and a hole , is given by the width of the momentum density matrix along the coordinate axes in figure si-2 ( supporting information ) . in this work , i arbitrarily define the coherence width as the distance where the momentum decreases to 10% of its maximum value . these figures show that , for a given number of benzene rings , the coherence size in the linear paraphenylenes is slightly larger than their cyclic counterparts . for the smaller paraphenylenes ( n < 9 ) , the coherence size in the linear systems is larger by approximately one repeat unit in comparison with the cyclic systems . as the number of benzene units increases to 18 , the coherence size for both the cyclic and the linear systems approach the same value of 9 repeat units . both |(q1)mn| and |(p1)mn| also show that the linear systems only have strong optical coherences induced at their center , whereas the optical coherences in the cyclic geometries are nearly equally distributed throughout the entire molecule . these different density - matrix delocalization patterns and coherence sizes result in distinct excitonic properties in the cyclic and linear systems , an effect which i quantify in the next section . to understand electronhole interactions on a more quantitative level , i calculated exciton binding energies for the cyclic and linear paraphenylenes as a function of size . as illustrated in figure 7 , the exciton binding energy , eexc , is given by the difference between the quasiparticle energy gap [ ionization potential ( ip ) electron affinity ( ea ) ] and the optical absorption gap ( eopt ) . both the ip and the ea were obtained from pbe0/6 - 31+g(d , p ) electronic energy calculations on the n 1 , n , and n + 1 electron systems at the neutral pbe0/6 - 31g(d , p ) optimized geometry . in this work , the vertical ip is defined by 3 and the magnitude of the vertical ea is given by 4 with these definitions , the magnitude of the exciton binding energy is then 5 table 2 gives ionization potentials , electron affinities , and exciton binding energies for the cyclic and linear paraphenylenes , and figure 8 shows the quasiparticle energy gap ( ip ea ) as a function of size . schematic representation of energy levels defining the exciton binding energy ( eexc ) , optical absorption gap ( eopt ) , ionization potential ( ip ) , and electron affinity ( ea ) . quasiparticle energy gaps ( ip ea ) for the cyclic and acyclic paraphenylenes as a function of the number of benzene rings . the quasiparticle energy gap for the acyclic paraphenylenes decreases much more rapidly than the energy gap for the corresponding cyclic systems . as figure 8 shows , with the exception of some small fluctuations , the quasiparticle energy gap in both systems decreases with size , as expected from quantum - confinement effects . furthermore , the quasiparticle energy gap for the linear paraphenylenes decreases much more rapidly than the energy gap for the corresponding cyclic systems . the question therefore arises : why does the quasiparticle energy gap behave so differently in the linear and cyclic systems ? to address this question , we must take a closer look at the variations in aromatic character within the linear and cyclic geometries . first , in each of these organic systems ( regardless of molecular geometry ) , an electronic competition exists between maintaining the aromaticity of the individual benzene rings versus delocalization along the backbone chain . in the unstrained linear paraphenylenes , there is relatively little conjugation along the backbone of the system because the electrons are localized in each individual phenyl unit to maintain aromaticity . in contrast , the cyclic paraphenylenes have highly strained geometries that distort the electronic structure of the individual phenyl units . the strong deformation within the phenyl ring diminishes the overlap of orbitals , resulting in quinoidal character ( antibonding interactions within the phenyl ring and double - bond character connecting adjacent phenyl rings ) . as a result , the electronic states in the cyclic paraphenylenes are more delocalized in comparison with their acyclic counterparts , in agreement with the transition density matrix analysis discussed in section . more importantly , the quinoid form is energetically less stable than the aromatic form : the quinoid structure has a smaller quasiparticle gap because it involves destruction of aromaticity and a loss in stabilization energy . in other words , by increasing the quinoid character of the system , the highest - occupied orbital in the cyclic paraphenylenes becomes destabilized ( i.e. , raised in energy ) , and the quasiparticle energy gap is significantly reduced . to give a quantitative measure of aromaticity in these systems , i calculated the nucleus - independent chemical shift ( nics(1 ) ) at the pbe0/6 - 31g(d , p ) level of theory for each of the phenyl rings in the cyclic and linear geometries . in the nics(1 ) procedure suggested by schleyer et al . , the absolute magnetic shielding is computed at 1 above and 1 below the phenyl ring center . the resulting average nics(1 ) values give a measure of -orbital aromaticity , with more negative nics(1 ) values denoting aromaticity and more positive values corresponding to quinoidal character . as a reference point in this work , the nics(1 ) value for benzene at the pbe0/6 - 31g(d , p ) table 2 gives average nics(1 ) values of phenyl rings in the cyclic and linear paraphenylenes , and figure 9 shows the nics(1 ) values as a function of size . as anticipated from our qualitative discussion on aromaticity , the nics(1 ) values for the cyclic paraphenylenes in particular , for the smaller nanorings ( n < 8) , figure 9 shows that these structures have significant quinoidal character , resulting in unusually small quasiparticle energy gaps ( cf . however , as the size of the cyclic paraphenylene increases , the strain energy becomes smaller , and both the cyclic and the linear nics(1 ) values approach the same limit . it is also interesting to note that the fluctuations in nanoring nics(1 ) values shown in figure 9 are correlated with the deviations seen in figures 4 and 8 . specifically , the discontinuous change in nics(1 ) values at n = 8 , 10 , 13 , and 17 can also be found as discontinuities in optical excitation energies ( figure 4 ) and abrupt changes in quasiparticle energy gaps in figure 8 . even more intriguing is the direct correlation between nics(1 ) values of individual phenyl rings and the intensity of the transition density matrix elements discussed in section . returning to figure 6 , the excitonic density along the diagonal for n = 18 is not uniform ; in other words , there are four areas along the diagonal with maximal density at repeat units of 3 , 8 , 12 , and 17 . figure si-3a , b ( supporting information ) displays the nics(1 ) values of individual phenyl rings and the dihedral angle between adjacent phenyl rings for the n = 18 cyclic geometry . figure si-3a ( supporting information ) shows four distinct maxima ( i.e. , four phenyl units that have quinoidal , or delocalized , character ) at the same positions corresponding to maxima in the n = 18 transition density matrix . in contrast , figure si-3b ( supporting information ) shows that the dihedral angles are nearly identical ( all within 0.3 ) throughout the n = 18 nanoring . taken together , these figures show that the distribution of excitonic density in figure 6 is purely an electronic effect and not a result of conformational variations . average nics(1 ) values of phenyl rings in the cyclic and acyclic paraphenylenes as a function of size . all nics(1 ) calculations were obtained at the pbe0/6 - 31g(d , p ) level of theory . finally , figure 10 displays the exciton binding energies of both systems calculated from eq 5 . in contrast to figures 4 and 8 , the exciton binding energies in both systems show a smooth monotonic variation as a function of size . as expected , for a given number of benzene rings , the exciton binding energies in the cyclic paraphenylenes are always larger ( and decrease at a faster rate ) than their acyclic counterparts . this trend is in agreement with the coherence sizes of the momentum density matrices discussed in section . compared with the linear paraphenylenes , the average electronhole distance in the cyclic systems is smaller , leading to an increase in the binding energy . however , as the number of benzene units increases , the coherence sizes in both systems become nearly equal , and the binding energies asymptotically approach the same value . these results , in combination with the quasiparticle energy gaps and electronhole delocalization patterns , explain the anomalous absorption energy trends in the cyclic paraphenylenes . in the linear systems , the quasiparticle energy gap ( ip ea ) decreases much more rapidly than the exciton binding energy , eexc . as a result , the behavior of the ip ea term dominates the right - hand side of eq 5 , and the optical absorption gap ( eopt = ip ea eexc ) in the linear paraphenylenes decreases as a function of size . in contrast , ip ea in the cyclic systems decreases at a significantly slower rate than eexc ( cf . consequently , eopt in the cyclic paraphenylenes will be largely determined by the behavior of eexc . because eexc decreases faster than ip ea in the carbon nanorings , the resulting optical absorption gap in the expression eopt = ip ea eexcincreases as a function of size , in agreement with the tddft calculations . exciton binding energies ( ip ea eopt ) for the cyclic and acyclic paraphenylenes as a function of the number of benzene rings . for a given number of benzene rings , in this study , i investigated the optoelectronic properties in a series of cycloparaphenylenes that form the molecular structure of carbon nanorings . to understand their unusual electronic properties as a function of molecular size , i analyzed tddft excited - state energies , examined electronhole transition density matrices , and calculated quasiparticle and exciton binding energies for both the cyclic and the linear paraphenylenes systems . the tddft calculations and transition density analyses show that the cyclic and linear paraphenylenes exhibit dramatically different size - dependent effects that can be understood in the framework of a simple exciton theory . specifically , the main differences between the absorption spectra of the cyclic and linear paraphenylenes are caused by the subtle interplay of contributions to the electronhole interaction energy . in the linear systems , the exciton binding energy decreases slower than the quasiparticle energy gap , and the resulting optical absorption gap decreases with molecular size , as expected . in contrast , the effective coulomb attraction between an electronhole pair is larger in the cyclic geometry and decreases at a faster rate than the quasiparticle band gap . as a result , the overall optical absorption gap in the cyclic paraphenylenes increases as a function of size . in conclusion , i have shown that the different excitonic effects in the cyclic and linear paraphenylenes play a vital role in determining their distinct photophysical properties . the unique spatial distributions of electronhole states , as depicted by their transition density delocalization patterns , result in different electronhole interaction energies that ultimately control the electronic excitations in these systems . looking forward , it would be very interesting to see if the unusual optical trends resulting from the lowest , weakly allowed transition in these nanorings can be observed spectroscopically . the most direct way to observe these lowest transitions would be via low - temperature spectroscopic studies with an applied magnetic field to brighten the dark excitons . a magnetic flux through the open center of the nanorings would distort the circular symmetry of the electronic wave function and produce bright excitons due to the aharonovbohm effect . it is also possible that one can already observe these lowest transitions spectroscopically without the use of an applied magnetic field . because these nanorings are significantly more flexible than carbon nanotubes , the lowest dark exciton may be strongly allowed through symmetry breaking . vibronic coupling , which allows a borrowing of intensity from an energetically close bright state , may also play a significant role in observing these weakly allowed transitions . for the larger carbon nanorings in particular , the first bright state ( i.e. , the s2 state ) is energetically close to the lowest dark state . as a result , the anomalous size scaling of optical excitations should be more easily observed in the larger carbon nanorings . from a theoretical point of view , it would also be interesting to see how these effects change ( or if they change at all ) when the paraphenylenes are functionalized with both electron donor and acceptor functional groups to facilitate charge transfer . calculations of this type would require the use of recent long - range - corrected density functional methods , which are currently underway in my group . with these goals in mind , i anticipate that a critical understanding of excitonic effects in molecular nanostructures will provide a step toward their implementation in optoelectronic applications .

a 52-year - old male patient presented to our hospital with a history of secondary hypertrophic osteoarthropathy ( hoa ) associated with an abdominal neoplasia and blepharoptosis . he had finger clubbing , hyperhidrosis , and hypertrichosis . he also had a recent history of extensive abdominal surgery with a pathology report of myelolipoma . routine blood work was unremarkable . upper eyelid reconstruction with blepharoplasty , upper eyelid wedge resection , and brow suspension was performed to address his eyelid concerns . by this case report , we would like to attract notice that the eyelid involvement may be a part of hoa and to emphasize the importance of systemic and pathologic evaluation in failed blepharoptosis surgery . the electronic structure and size - scaling of optoelectronic properties in cycloparaphenylene carbon nanorings are investigated using time - dependent density functional theory ( tddft ) . the tddft calculations on these molecular nanostructures indicate that the lowest excitation energy surprisingly becomes larger as the carbon nanoring size is increased , in contradiction with typical quantum confinement effects . in order to understand their unusual electronic properties , i performed an extensive investigation of excitonic effects by analyzing electron - hole transition density matrices and exciton binding energies as a function of size in these nanoring systems . the transition density matrices allow a global view of electronic coherence during an electronic excitation , and the exciton binding energies give a quantitative measure of electron - hole interaction energies in the nanorings . based on overall trends in exciton binding energies and their spatial delocalization , i find that excitonic effects play a vital role in understanding the unique photoinduced dynamics in these carbon nanoring systems .

osteoporosis is defined as a skeletal disorder characterized by loss of bone mass , decreased bone strength , and increased risk of bone fracture.1 the disease progresses with age , especially in postmenopausal women.2 japan is one of the most rapidly aging societies worldwide . according to the national institute of population and social security research ( tokyo , japan ) , japanese individuals over 65 years of age constitute 23.0% of the population in 2010 and 25.1% to 25.2% as of 2013 , meaning that more than one in four people in japan are elderly . according to population - based epidemiologic studies , the estimated number of people with osteoporosis in japan is 13 million , among which 130,000 suffer from hip fractures every year ; 20,000 of these individuals die and 60,000 experience functional decline . the prevalence rate of vertebral fractures in japan is reportedly comparable with or higher than in caucasian populations , and is approximately 30% in women in their 70 s and 40% in those in their 80 s.3 a study of the prevalence of vertebral fractures in hong kong , thailand , indonesia , and japan revealed that the prevalence of vertebral fractures in both men and women was highest in japan for the younger ( 6574 years ) and older ( 75 years ) age groups ( 36.6% and 37.6% for men , 18.8% and 28.7% for women , respectively).4 bisphosphonates are known to increase bone mineral density by inhibiting osteoclast - mediated bone resorption and thereby reduce the risk of fractures.5 several formulations containing bisphosphonates have been applied to the management of osteoporosis . alendronate sodium hydrate ( alendronate ) is a bisphosphonate that potently inhibits bone resorption and is used for the treatment of osteoporosis . alendronate produces a sustained reduction in the levels of biochemical markers of bone remodeling , returning them to the premenopausal range.6,7 it also increases bone mineral density612 and decreases the risk of osteoporotic fracture in postmenopausal women.812 long - term intervention studies have shown that continuous alendronate therapy is associated with a sustained therapeutic effect on bone density and remodeling.13,14 however , bisphosphonates including alendronate , suppress bone remodeling and thus may prevent the repair of microdamage . nine patients reportedly developed spontaneous nonspinal fractures after 1 to 8 years of alendronate therapy , and histomorphometric analysis of the bone biopsy samples revealed severe suppression of bone turnover.15 in japan , a patient with insufficiency fractures of the pubis and ilium related to severe suppression of bone turnover after nearly 3 years of alendronate therapy was reported.16 recent papers reported side effects of both atypical femoral fractures and osteonecrosis of the jaw after 7 years of oral alendronate therapy.17 bisphosphonates also have adverse gastrointestinal effects , such as erosions and ulcers in the stomach and small intestine , which are mediated by mitochondrial superoxide production and lipid peroxidation.18 alendronate oral tablets were produced and are used for the treatment of osteoporosis . water is required with an alendronate oral tablet to help facilitate absorption , and the drug can not be taken with other foods or drinks . taking a tablet with oral jelly is a method to prevent digestive system disease and reduce the choking hazard . in this review , this new formulation of alendronate sodium hydrate ( oral jelly ) is introduced and discussed in the treatment of osteoporosis . in addition to osteoporosis , many elderly persons suffer from cardiovascular disease , diabetes mellitus , and joint disease . to treat their diseases , they often use multiple oral drugs . ease of swallowing and handling are preferable for medications , particularly those for elderly patients . studies on the construction of new pharmaceutical preparations and new packaging containers that are optimal for administration to elderly people conducted by the ministry of health and welfare of japan in the 1980s reported that most powder , capsule , tablet , and liquid drug formulations and most packaging containers were suitable for general healthy adults , but not for elderly persons.19 development of new formulations of medicines suitable for elderly individuals with difficulty in swallowing and mastication , dry mouth , and artificial teeth was desired . in 1996 , the ministry of health and welfare of japan reported that jelly , pudding - like , biscuit - like , and wafer - like formulations were preferable for elderly patients.20 biscuit - like and wafer - like formulations were developed as orally disintegrating tablets , which were advanced and popular drug formulations.21 the oral jelly formulation was developed as an original formulation in japan.22 oral jelly formulations have many advantages : ease of swallowing , the ability to take the medication without water , lower risk of accidental ingestion , and optimal masking of drug taste.22 in addition to drug formulations , packaging containers optimal for elderly individuals were developed in japan . there are several types of packaging containers for oral jelly : a single - serving type ( figure 1 ) , a stick type ( figure 2 ) , and an air - extruded type ( table 2 ) . according to a survey among individuals aged 65 and over , the air - extruded type was better than the single - serving type and the stick type.23 elderly persons tend to suffer from a decline in physical performance . it is difficult for the elderly persons to handle the single - serving type when it involved peeling off the container s seal and taking the jelly formulation with a spoon . for the stick type , it was difficult to take all of the jelly formulation by squeezing the packaging container . an air - extruded jelly formulation was developed as a new formulation comprising the jelly formulation and clean air under increased pressure.2225 the jelly formulation is smoothly discharged by pushing the air portion . therefore , elderly patients with physical disabilities are able to easily take all of the jelly formulation from the package . the questionnaire survey indicated the applicability of the air - extruded jelly formulation in elderly patients.26 a majority of the patients ( 63.9% , 69 of 108 subjects ) intended to switch their present drug formulation to the air - extruded jelly formulation . especially among subjects who presently took a powder formulation or more than five kinds of medications daily , a high ratio intended to switch to the air - extruded jelly formulation . alendronate oral jelly 35 mg ( bonalon oral jelly 35 mg ; teijin pharma limited , tokyo , japan ) was approved in 2012 by the ministry of health , labour and welfare of japan as the world s first drug for osteoporosis in jelly formulation . alendronate oral jelly 35 mg , administered once a week , was launched in 2012 ( figure 3 ) . once - weekly alendronate oral jelly comprises a jelly portion and an air portion , which features the air - extruded jelly formulation and makes it easy to take the jelly formulation when pushed by the air . thus , the active ingredient in the once - weekly alendronate oral tablet 35 mg , which has been proven both safe and effective as an osteoporosis treatment , is offered in a jelly formulation . a dissolution test and bioequivalence study , in accordance with the japanese guideline on bioequivalence assessment for development of additional formulations and the japanese guideline for bioequivalence studies of generic products , were carried out to evaluate the bioequivalence of alendronate oral jelly 35 mg to the alendronate oral tablet 35 mg.27 in the dissolution test , the dissolution profiles of both formulations satisfied the criteria for judgment of similarities set forth in the guideline . the bioequivalence study was carried out with an open - label , randomized cross - over ( two - sequence , two - period ) study design and included 32 japanese healthy adult male volunteers . when the pharmacokinetic parameters of alendronate oral jelly 35 mg and alendronate oral tablet 35 mg were calculated from the serum alendronic acid concentration , the area under the blood concentration time curve ( auc ) showed a mean standard deviation of 21.70 10.20 ng hour / ml and 21.33 13.96 ng hour / ml , and maximum concentration ( cmax ) was 9.60 4.36 ng / ml and 9.03 4.93 ng / ml , respectively ( table 3 ) . the serum half - life ( t1/2 ) was 1.54 0.48 hours and 1.47 0.50 hours , respectively . the mean logarithmic auc and cmax were log ( 1.049 ) and log ( 1.050 ) , respectively , each within the criteria range , log ( 0.90 ) to log ( 1.11 ) , for judgment of bioequivalence with the similarities in dissolution profiles . these results indicate that alendronate oral jelly 35 mg is bioequivalent to alendronate oral tablet 35 mg and suggest no difference in clinical efficacy and safety . in european and american studies , alendronate at a dose of 5 to 20 mg / day has consistently been shown to increase bone mineral density at the spine , hip , and other skeletal sites and to reduce the risk of all types of fractures.612,2832 the fracture intervention trial ( fit ) comprised two independent but parallel studies with common recruitment conducted at 11 institutions in the united states . the alendronate dosage was 5 mg / day for 2 years , and was then increased to 10 mg / day.8,10 in the phase iii study , alendronate dosages ranged from 5 to 20 mg / day.9 in the fosamax international trial ( fosit ) and the long - term care facilities study ( ltcfs ) , the alendronate dosage was 10 mg / day . compared to caucasians , asians have a smaller body size33 and a lower dietary intake of calcium.34 because of these racial differences , the recommended drug dose established for caucasians may not be appropriate for asians . a small - scale , single - blind study of postmenopausal japanese women indicated that a dosage of 2.5 mg / day was as effective as a dosage of 10 mg / day in increasing lumbar bone mineral density with 36 weeks of treatment . however , bone turnover markers changed more markedly and rapidly in the 10 mg / day group , and the incidence of adverse effects was higher in the 10 mg / day group . these findings suggest that a 10 mg / day dosage may be an overdosage for japanese patients.35 a dose - range finding study in japan concluded that the optimal daily dose of alendronate is 5 mg in a japanese population.36 the study showed that alendronate at a dosage of 5 mg / day was effective in increasing lumbar bone mineral density , and the incidence of adverse effects was no different compared with the placebo control group after 24 weeks of treatment for japanese patients with postmenopausal osteoporosis . the study also showed that serum parathyroid hormone and serum 1,25-dihydroxyvitamin d increased only in the 5 mg / day dose group . studies to evaluate the effects of alendronate for the treatment of japanese patients have been carried out . a double - masked multicenter comparative study of japanese patients with osteoporosis indicated that alendronate treatment at a dosage of 5 mg / day for 48 weeks was more efficacious than alfacalcidol for increasing bone mineral density without serious drug - related adverse events.37 a 2 year , double - blind comparative study of japanese patients with osteoporosis indicated that alendronate at a dosage of 5 mg / day significantly lowered the incidence of one or more vertebral fractures to a greater extent than did alfacalcidol after 6 months or more of treatment.38 a consecutive 3 year follow - up study of japanese patients demonstrated that continuous alendronate therapy at 5 mg / day is associated with a sustained therapeutic effect on increasing vertebral bone mineral density and reducing the risk of new vertebral fractures.39 a prospective study to assess vertebral strength and mechanical parameters in japanese women with postmenopausal osteoporosis indicated that vertebral strength significantly increased by 26.9% from baseline at 12 months of 5 mg / day alendronate,40 and by 31.8% at 18 months,41 demonstrating a sustained therapeutic effect . once - daily treatment with alendronate has been shown to substantially increase bone mineral density and bone strength and to reduce the incidence of osteoporotic fractures . dosing convenience is a key element in the effective management of any chronic disease , and is particularly important in the long - term management of osteoporosis . alendronate must be administered after fasting with water only , at least 30 minutes before the first food of the day , which offers an additional degree of complexity . thus , it is desirable to reduce the frequency of alendronate dosing without compromising treatment efficacy . animal data support the rationale that once - weekly dosing with alendronate at 70 mg ( seven times the daily oral treatment dose for caucasians ) could provide similar efficacy to that of alendronate at a dosage of 10 mg / day because of its long duration of effect in bone.4244 in addition , animal studies have suggested that the potential for esophageal irritation , observed with daily oral alendronate , might be substantially reduced with once - weekly dosing.45,46 a randomized , double - blind , multicenter study to evaluate the efficacy and safety of oral alendronate once- and twice - weekly dosing regimens compared with daily dosing in postmenopausal women with osteoporosis showed that both the once- and twice - weekly dosing regimens fully satisfied the equivalence criteria relative to daily dosing therapy.47,48 this study also showed that there were fewer serious upper gastrointestinal adverse experiences and a trend toward a lower incidence of esophageal events in the once - weekly dosing group than in the daily dosing group . the study concluded that once - weekly treatment with alendronate would provide patients with a more convenient , therapeutically equivalent alternative to daily dosing and might enhance compliance and long - term persistence with therapy . a therapeutic equivalence study of once - weekly and once - daily alendronate was also carried out in japan . the efficacy and safety of treatment with once - weekly oral alendronate 35 mg were compared with those of once - daily oral alendronate 5 mg in a 52-week , randomized , double - blind , multicenter study of japanese patients with osteoporosis.49 the study concluded that the efficacy and safety of the alendronate 35 mg once - weekly regimen were therapeutically equivalent to those of the alendronate 5 mg once - daily regimen . for a 12 month period in the united states , a randomized and double - blind study to compare the efficacy and tolerability of once - weekly oral alendronate 70 mg and the once - daily oral selective estrogen receptor modular raloxifene 60 mg was carried out for the treatment of postmenopausal osteoporosis.50 the study concluded that the once - weekly alendronate 70 mg regimen produced significantly greater increases in spine and hip bone mineral density and greater reductions in markers of bone turnover than the once - daily raloxifene 60 mg regimen with similar safety and tolerability profiles . risedronate is a bisphosphonate that , like alendronate , binds to bone hydroxyapatite and inhibits osteoclast - mediated bone resorption . once - weekly risedronate oral tablets at a dosage of 35 mg / week ( 5 mg / day ) have been produced and used to treat osteoporosis . a study comparing the once - weekly alendronate 70 mg regimen with the once - weekly risedronate 35 mg regimen during 24 months showed that the former yielded significantly greater bone mineral density gains and larger decreases in bone turnover marker levels than the latter , with no difference in gastrointestinal tolerability.51 the us food and drug administration approved the once - monthly oral ibandronate sodium tablet as the first once - monthly oral tablet in 2005 . ibandronate sodium is a bisphosphonate that inhibits bone resorption and is commonly prescribed for the treatment and prevention of postmenopausal osteoporosis.52,53 once - weekly oral bisphosphonates have been associated with higher adherence compared with the once - daily formulations , although overall adherence has remained suboptimal.5456 a study performed to assess whether once - monthly bisphosphonates are associated with improvements in adherence showed no evidence of improved adherence with once - monthly dosing relative to once - weekly dosing , although adherence with either weekly or monthly dosing was significantly better than that with once - daily dosing.57 these study results suggests that merely reducing the dosing frequency of oral bisphosphonates is not enough to improve adherence . alendronate oral jelly , which features the air - extruded jelly formulation , is easy for elderly patients to use and take ; most of these patients have physical disabilities and swallowing difficulty . therefore , this new formulation of osteoporosis treatment may meet individual needs and improve adherence . in addition , alendronate oral jelly with its advantages of ease of swallowing and lower risk of accidental ingestion may reduce the incidence of adverse effects such as esophageal irritation first , there are no published clinical data for its use in the treatment of osteoporosis . an in vivo study to evaluate the use of alendronate sodium gel in the treatment of bone resorptive lesions in periodontitis was carried out.58 osseous defects associated with chronic adult periodontitis were filled with alendronate sodium gel , and flaps were then sutured to obtain primary closures of the interdental papilla in the alendronate gel site . in the control site the osseous defect improvement rate in the alendronate gel site after 6 months was 42.85% and showed significant improvement compared with that of the control site of ( 7.43% ) . in addition , a 6 month , randomized , controlled clinical trial showed that the local delivery of 1% alendronate gel into periodontal pockets significantly improved bone filling compared with a placebo gel.59 second , alendronate oral jelly is approved and used only in japan . third , once - weekly dosing of alendronate oral jelly is the only dosing regimen . other options , such as a once - monthly dosing regimen , will create a variety of osteoporosis treatments to meet individual needs . intravenous infusion of alendronate for the treatment of osteoporosis is one of these therapeutic options . this preparation may avoid the topical effects of oral alendronate on the gastric mucosa and may provide a useful alternative to oral dosing . a 52-week , multicenter , randomized , double - masked , active - controlled , parallel - group , non - inferiority study of different preparations of alendronate in japanese patients with osteoporosis , randomly assigned to receive intravenous alendronate 900 g every 4 weeks or oral alendronate 35 mg once weekly was conducted.60 the study confirmed that the efficacy and safety of intravenous alendronate 900 g every 4 weeks were similar to those of oral alendronate 35 mg taken once weekly , although the study did not show that intravenous administration might reduce the incidence of gastrointestinal adverse effects . an intravenous infusion of alendronate will provide a new opportunity for effective treatment of patients with osteoporosis who are unable to take oral alendronate or have difficulty complying with the procedures for oral dosing . these new formulations of alendronate oral jelly and intravenous alendronate provide treatment alternatives so that patients may select a method of dosing tailored to their preferences . management of osteoporosis involves assessing fracture risk and preventing fractures.61 higher adherence of treatment of patients with osteoporosis and prevention of fractures associated with osteoporosis remain issues to be resolved . specific developmental disturbances due to various insults , brain damage , toxic influences , or psychological stress may determine higher levels of neurocognitive integration dysfunction that typically occur in children with attention - deficit and hyperactivity disorder ( adhd).13 one of the most important postnatal developmental deficits which may have various etiological causes influencing higher motor and cognitive functions is the persistence of several primitive reflexes that occurs in some neuropsychiatric disorders , such as the symmetric tonic neck reflex ( stnr ) and asymmetric tonic neck reflex ( atnr).46 these findings are in agreement with basic neurological concepts discovered by hughlings jackson , who proposed that certain functions developed later during the ontogenesis of the central nervous system ( cns ) tend to replace older ones.7 although hughlings jackson mainly focused on epilepsy in his works , he later discovered certain basic developmental rules describing cns development that were characterized by hierarchical ontogenetic stages.8 when the higher stages of cns development are not successfully achieved or are damaged , lower neural functions may be disinhibited and their release from control may lead to the dysregulation of later - developed adaptive functions.7 in principle , these findings agree with data indicating hypofrontality in adhd,9,10 which suggests that attentional and executive dysfunctions related to reduced activation in the right inferior frontal cortex , anterior cingulate cortex , supplementary motor area , and other regions are linked to inhibitory deficits.3 according to current findings and the best of our knowledge , there are few reported findings of persisting reflexes in dyslexia11,12 and preliminary findings suggesting that adhd symptoms could be related to persistence of the primitive reflexes in later developmental stages than is usual.12,13 these studies suggest that further detailed research examining developmental conditions in adhd is warranted and that such research might have significant consequences for understanding adhd , dyslexia , and other neurodevelopmental disorders.1113 most recent studies examining atypical development in adhd have predominantly studied boys , while studies of girls are very rare . however , the studies examining developing children have shown specific patterns mainly characterized by the girls earlier brain maturation compared with that of boys.14 at this time , as far as we are aware , there is no reported study that has assessed whether adhd symptoms are related to persistence of the primitive reflexes in medication - nave children with a focus on sex - specific assessment . as such , in the study reported here we examined the extent to which the persistence of the primitive atnr and stnr is related to adhd symptoms in 35 medication - nave girls of school age ( 811 years ) . data on 35 girls ( mean age 9.51 , standard deviation [ sd ] = 1.01 , age range 811 years ) with adhd diagnosed according to diagnostic and statistical manual of mental disorders : fourth edition ( dsm - iv ) criteria characterizing their primitive reflexes and psychometric measures of adhd symptoms were collected . all the patients were medication nave and their assessment was done before starting any treatment . the adhd diagnosis was combined hyperactive - inattentive subtype with occurrence of adhd - based learning difficulties and , in a few cases , episodic enuresis . exclusion criteria were other child psychiatric and neurological disorders , including conduct disorder , alcohol or drug dependency , substance abuse , and/or previous exposure to stimulant medication . recruitment was based on consecutive visits of the untreated patients to the clinic , and the control group was recruited using school advertisements . the control group of 30 girls without adhd ( mean age 9.83 , sd = 0.98 , age range 811 years ) fulfilled the same exclusion criteria . parents of all the participants gave written informed consent and the research was approved by the charles university first faculty of medicine ethical committee within the framework of collaboration with the elspac project , the project of the center for neuropsychiatric research of traumatic stress , and the msm0021620849 project at charles university . the measurement of primitive reflexes was done twice by two independent examiners and the results of the two measurements averaged . we measured atnr using the schilder test.15 atnr presents as the tonic reflex response that occurs in newborns ; it normally vanishes at around 3 months of age . in the schilder test , the subject stands with their feet together and the arms held straight out at shoulder level and height but with the hands relaxed at the wrists . in the test when i turn your head , i want you keep your arms straight out in front of you , as they are now . this means your arms remain in the same position , and only your head moves . then the tester slowly rotates the subject s head until the chin is parallel with the shoulder , pauses for 10 seconds , then returns the head to the midline and again pauses for 10 seconds . typical indicators of the atnr include movement of the extended arms in the same direction as the head turn , dropping of the arms , or swaying and loss of balance . it was scored as follows : 0 = no response ( the arms remained straight out in front ) ; 1 = slight movement of the arms up to 20 to the same side as the head is turned or slight dropping of the arms ; 2 = movement of the arms up to 45 as the head is turned or marked dropping of the arms ; 3 = arm movement greater than 45 either to the side or down , swaying or loss of balance may occur . purdue reflex test.16 this reflex emerges at 68 months of life and is inhibited at between 9 and 11 months . in the bender purdue test , the subject is instructed to maintain four point kneeling table position and to slowly move the head to look down as between the thighs . the position is held for up to 5 seconds and then the head is slowly moved upward as if looking at the ceiling . typical indicators of the reflex are any bending of the arms due to head flexion and/or raising of the feet , straightening of the arms and flexion of the knees as a result of head extension , any bending of the arms due to head flexion and/or raising of the feet , and straightening of the arms and flexion of the knees due to head extension . the test was scored as follows : 0 = no response ; 1 = tremor in one or both arms or slight hip movement ; 2 = movement of the elbow on either side and/or definite , movement in the hips , or arching of the back ; 3 = bending of the arms on head flexion or movement of the bottom back on head extension ; 4 = bending of the arms to the floor , or movement of the bottom back onto the ankles , so that the subject is sitting in the cat position . a frequently used measure of adhd symptoms is the conners parent questionnaire ( cpq).17 the cpq is 93-item scale of symptoms that are most commonly associated with behavioral disorders and related to children and adolescents ( aged 317 years ) , and can measure treatment changes and outcome assessment purposes . the questionnaire enables a total score to be calculated as well as scores for subscales based on the factor structure of the questionnaire : i conduct problems ( items 39 , 40 , 41 , 47 , 48 , 51 , and 69 ) ; ii anxiety ( items 811 , 42 , 43 , and 64 ) ; iii impulsivity - hyperactivity ( items 78 , 8084 , 89 , and 90 ) ; iv learning problems ( items 45 , 62 , 63 , 67 ) ; v psychosomatic difficulties ( items 6 and 2124 ) ; vi perfectionism ( items 3 and 7678 ) ; vii antisocial behavior ( items 7173 and 75 ) ; and viii muscular tension ( items 1214 and 36 ) . the symptoms are rated on a four - point likert scale ( from 0 to 3 ) by either one or both parents of the child . for statistical evaluation of scores of the measures of atnr , stnr , and adhd symptoms , nonparametric analyses were preferred because cpq , atnr , and stnr data do not have normal distribution . all the methods of statistical evaluation were performed using the software package statistica ( v 6 ; statsoft inc , tulsa , ok , usa ) . data on 35 girls ( mean age 9.51 , standard deviation [ sd ] = 1.01 , age range 811 years ) with adhd diagnosed according to diagnostic and statistical manual of mental disorders : fourth edition ( dsm - iv ) criteria characterizing their primitive reflexes and psychometric measures of adhd symptoms were collected . all the patients were medication nave and their assessment was done before starting any treatment . the adhd diagnosis was combined hyperactive - inattentive subtype with occurrence of adhd - based learning difficulties and , in a few cases , episodic enuresis . exclusion criteria were other child psychiatric and neurological disorders , including conduct disorder , alcohol or drug dependency , substance abuse , and/or previous exposure to stimulant medication . recruitment was based on consecutive visits of the untreated patients to the clinic , and the control group was recruited using school advertisements . the control group of 30 girls without adhd ( mean age 9.83 , sd = 0.98 , age range 811 years ) fulfilled the same exclusion criteria . parents of all the participants gave written informed consent and the research was approved by the charles university first faculty of medicine ethical committee within the framework of collaboration with the elspac project , the project of the center for neuropsychiatric research of traumatic stress , and the msm0021620849 project at charles university . the measurement of primitive reflexes was done twice by two independent examiners and the results of the two measurements averaged . we measured atnr using the schilder test.15 atnr presents as the tonic reflex response that occurs in newborns ; it normally vanishes at around 3 months of age . in the schilder test , the subject stands with their feet together and the arms held straight out at shoulder level and height but with the hands relaxed at the wrists . in the test when i turn your head , i want you keep your arms straight out in front of you , as they are now . this means your arms remain in the same position , and only your head moves . then the tester slowly rotates the subject s head until the chin is parallel with the shoulder , pauses for 10 seconds , then returns the head to the midline and again pauses for 10 seconds . typical indicators of the atnr include movement of the extended arms in the same direction as the head turn , dropping of the arms , or swaying and loss of balance . it was scored as follows : 0 = no response ( the arms remained straight out in front ) ; 1 = slight movement of the arms up to 20 to the same side as the head is turned or slight dropping of the arms ; 2 = movement of the arms up to 45 as the head is turned or marked dropping of the arms ; 3 = arm movement greater than 45 either to the side or down , swaying or loss of balance may occur . purdue reflex test.16 this reflex emerges at 68 months of life and is inhibited at between 9 and 11 months . in the bender purdue test , the subject is instructed to maintain four point kneeling table position and to slowly move the head to look down as between the thighs . the position is held for up to 5 seconds and then the head is slowly moved upward as if looking at the ceiling . typical indicators of the reflex are any bending of the arms due to head flexion and/or raising of the feet , straightening of the arms and flexion of the knees as a result of head extension , any bending of the arms due to head flexion and/or raising of the feet , and straightening of the arms and flexion of the knees due to head extension . the test was scored as follows : 0 = no response ; 1 = tremor in one or both arms or slight hip movement ; 2 = movement of the elbow on either side and/or definite , movement in the hips , or arching of the back ; 3 = bending of the arms on head flexion or movement of the bottom back on head extension ; 4 = bending of the arms to the floor , or movement of the bottom back onto the ankles , so that the subject is sitting in the cat position . we measured atnr using the schilder test.15 atnr presents as the tonic reflex response that occurs in newborns ; it normally vanishes at around 3 months of age . in the schilder test , the subject stands with their feet together and the arms held straight out at shoulder level and height but with the hands relaxed at the wrists . in the test when i turn your head , i want you keep your arms straight out in front of you , as they are now . this means your arms remain in the same position , and only your head moves . then the tester slowly rotates the subject s head until the chin is parallel with the shoulder , pauses for 10 seconds , then returns the head to the midline and again pauses for 10 seconds . typical indicators of the atnr include movement of the extended arms in the same direction as the head turn , dropping of the arms , or swaying and loss of balance . it was scored as follows : 0 = no response ( the arms remained straight out in front ) ; 1 = slight movement of the arms up to 20 to the same side as the head is turned or slight dropping of the arms ; 2 = movement of the arms up to 45 as the head is turned or marked dropping of the arms ; 3 = arm movement greater than 45 either to the side or down , swaying or loss of balance may occur . purdue reflex test.16 this reflex emerges at 68 months of life and is inhibited at between 9 and 11 months . in the bender purdue test , the subject is instructed to maintain four point kneeling table position and to slowly move the head to look down as between the thighs . the position is held for up to 5 seconds and then the head is slowly moved upward as if looking at the ceiling . typical indicators of the reflex are any bending of the arms due to head flexion and/or raising of the feet , straightening of the arms and flexion of the knees as a result of head extension , any bending of the arms due to head flexion and/or raising of the feet , and straightening of the arms and flexion of the knees due to head extension . the test was scored as follows : 0 = no response ; 1 = tremor in one or both arms or slight hip movement ; 2 = movement of the elbow on either side and/or definite , movement in the hips , or arching of the back ; 3 = bending of the arms on head flexion or movement of the bottom back on head extension ; 4 = bending of the arms to the floor , or movement of the bottom back onto the ankles , so that the subject is sitting in the cat position . a frequently used measure of adhd symptoms is the conners parent questionnaire ( cpq).17 the cpq is 93-item scale of symptoms that are most commonly associated with behavioral disorders and related to children and adolescents ( aged 317 years ) , and can measure treatment changes and outcome assessment purposes . the questionnaire enables a total score to be calculated as well as scores for subscales based on the factor structure of the questionnaire : i conduct problems ( items 39 , 40 , 41 , 47 , 48 , 51 , and 69 ) ; ii anxiety ( items 811 , 42 , 43 , and 64 ) ; iii impulsivity - hyperactivity ( items 78 , 8084 , 89 , and 90 ) ; iv learning problems ( items 45 , 62 , 63 , 67 ) ; v psychosomatic difficulties ( items 6 and 2124 ) ; vi perfectionism ( items 3 and 7678 ) ; vii antisocial behavior ( items 7173 and 75 ) ; and viii muscular tension ( items 1214 and 36 ) . the symptoms are rated on a four - point likert scale ( from 0 to 3 ) by either one or both parents of the child . for statistical evaluation of scores of the measures of atnr , stnr , and adhd symptoms , spearman correlation coefficients were determined and multiple linear regression analysis was undertaken . nonparametric analyses were preferred because cpq , atnr , and stnr data do not have normal distribution . all the methods of statistical evaluation were performed using the software package statistica ( v 6 ; statsoft inc , tulsa , ok , usa ) . adhd symptoms measured by total score of the cpq and its subscales were significantly correlated with atnr and stnr scores ; that is : cpq and atnr , r = 0.64 ; cpq and stnr , r = 0.41 ; ii anxiety and atnr , r = 0.39 ; iii impulsivity - hyperactivity and atnr , r = 0.47 ; iii impulsivity - hyperactivity and stnr , r = 0.40 ; iv learning problems and stnr , r = 0.47 ; and vi perfectionism and atnr , r = 0.44 ( table 1 , figure 1 ) . spearman rank correlation coefficients with r higher than 0.43 were significant at p < 0.01 , with fisher s z > 0.46 , and spearman rank correlation coefficients with r > 0.33 significant at p < 0.05 , with fisher s z > 0.35 . these correlations indicate that significant manifestations of adhd symptoms are associated with persisting atnr and stnr responses . we compared cpq , atnr and stnr scores for the 35 study subjects with those for the 30 control participants . the control group had no adhd symptoms , and none of this group manifested atnr ( the score was zero in all cases ) , while only one case of stnr response was found with a score of 1 . to analyze effects of atnr and stnr on cpq , we used a multiple linear regression to determine whether persisting primitive reflexes in their specific interactions are proportionally linked to increased levels of cpq . multiple r = 0.60 was found to be statistically significant ( p = 0.0008 ; f = 8.99 ; sequential consistency ( sc ) model = 2,375 ; sc residuals ( resid ) = 4,229 ; processor consistency ( pc ) model = 1187 ; pc resid = 132 ) which enables cpq to be defined as a linear function of two variables : cpq = f(atnr , stnr ) . recent findings show that a high proportion of children with adhd exhibit motor abnormalities.3,4 according to brain imaging studies , these abnormalities are probably linked to prefrontal cortex deficits that influences attention and executive functions.1820 the abnormalities may also be of cerebellar origin and adhd children in many cases exhibit atrophy in cerebellar regions associated with motor dysfunctions that are most likely linked to inhibitory deficits.3,21,22 disinhibitory processes related to disturbed motor functions are probably also linked to persisting primitive reflexes that were not sufficiently suppressed in later stages of development . according to some evidence , persisting primitive reflexes may occur in various neuropsychiatric syndromes such as schizophrenia or various forms of dementia5,6,20,23 and have also been observed in patients with dyslexia11,24 and in school - aged children with adhd12,13,25,26 in our previous study on medicated adhd children,13 we found that boys had higher levels of adhd symptoms and that there was little statistically significant difference in persisting primitive atnr between girls and boys . moreover , studies that have focused on persistent atnr in dyslexia11,24 have not reported differences between girls and boys . in this context , this study represents an important step focused on detailed analysis of adhd children in medication nave conditions . the results show that persistent atnr and other primitive reflexes that may occur in later age specifically influence adhd symptoms related to hyperactivity due to muscular tension , which also may have specific influence on attentional functions . taken together recent research predominantly does not take into account differences between adhd girls and boys.27,28 nevertheless several comparisons showed few sex effects in core clinical manifestations of adhd . in addition , girls compared with boys , have significantly more parent - rated emotional symptoms and more likely they become victims of bullying . girls and boys have similar levels of coexisting psychiatric and physical health problems , and receive the same types of treatment although fewer girls than boys are referred for adhd treatment.27,28 therefore , research focused on girls with adhd seems to be particularly important and , in the future , detailed comparison of girls and boys may provide particularly important findings and have significant therapeutic implications . the results of this study support the hypothesis that adhd symptoms are linked to the persisting primitive atnr and stnr in girls . further data are needed to find specific sex differences and to determine the relationships between adhd symptoms and various primitive reflexes that are possible to assess in school - aged children . together , our data suggest a hypothesis for future research that adhd symptoms may present a process related to primitive neural mechanisms , such as primitive reflexes , interfering with higher - level brain functions due to insufficiently developed cognitive and motor integration . in this context , hughlings jackson s concept may in principle be useful to explain and understand certain developmental changes as a consequence of disturbances between higher and lower levels of cognitive and motor functions during brain processing that might cause processes related to dissolution ( or disintegration ) of mental functions.8,12 the preliminary data reported here show that detailed analysis of persisting primitive reflexes in children with developmental disorders and comparisons of differences between girls and boys with adhd and dyslexia may offer important new findings with specific consequences for treatment .

osteoporosis is a skeletal disorder characterized by loss of bone mass , decreased bone strength , and an increased risk of bone fracture . the disease progresses with age , especially in postmenopausal women . japan is one of the most rapidly aging societies worldwide . japanese individuals over 65 years of age constituted 23.0% of the population in 2010 and 25.1% to 25.2% as of 2013 . the estimated number of people with osteoporosis in japan is currently 13 million . bisphosphonates increase bone mineral density by inhibiting osteoclast - mediated bone resorption , thereby reducing the risk of fractures . alendronate sodium hydrate ( alendronate ) is a bisphosphonate that potently inhibits bone resorption and is used to treat osteoporosis . sufficient water is required to take an alendronate oral tablet ; insufficient water could result in digestive system diseases , such as esophageal ulceration . elderly patients with swallowing difficulty may choke on the tablet . taking a tablet with oral jelly is a method to prevent digestive system disease and reduce the choking hazard . once - weekly alendronate oral jelly was approved in 2012 by the ministry of health , labour , and welfare of japan as the world s first drug for osteoporosis in a jelly formulation . it consists of a jelly portion and an air portion . the jelly formulation is smoothly discharged by pushing the air portion . therefore , elderly patients with physical disabilities are able to easily take all of the jelly formulation from the package . in this review , this new formulation of alendronate sodium hydrate ( oral jelly ) is introduced and discussed in terms of osteoporosis treatment . this new formulation provides an alternative so that patients may select a method of dosing tailored to their preferences . management of osteoporosis involves assessing fracture risk and preventing fractures . higher adherence to the treatment of patients with osteoporosis and prevention of osteoporotic fractures are issues to be resolved . background and objectivesrecent and historical findings suggest that later - developed functions during brain ontogenesis related to higher levels of cognitive and motor integration tend to replace the older , more primitive , ones , and the persistence of the older functions may be linked to specific neuropsychiatric disorders . currently , there is growing evidence to suggest that persisting primitive reflexes may be related to developmental and neurodegenerative disorders . preliminary data also suggest that persisting primitive reflexes may be specifically linked to attention - deficit and hyperactivity disorder ( adhd).methodsin the study reported here , we tested to what extent the persisting primitive asymmetric tonic neck reflex and symmetric tonic neck reflex are related to adhd symptoms measured by conners parent questionnaire in 35 medication - nave girls of school age ( 811 years ) with adhd . the results were compared with those of a control group of 30 girls of the same age.resultsthis study showed that persisting primitive reflexes are closely linked to adhd symptoms.conclusionthe data suggest that adhd symptoms may be linked to more primitive neural mechanisms interfering with higher brain functions due to insufficiently developed cognitive and motor integration .

community - acquired pneumonia ( cap ) is acquired during the course of everyday life by patients who do not have a known immune deficiency . despite advancements in antibiotic treatment , cap continues to negatively affect morbidity and mortality . cap is also the sixth most common infectious cause of death in hospitals.1 previous studies have shown that the mortality rate of pneumonia varies from 1% to 60% and is related to the weight of the patient.2 studies have found that the mortality rate in cap outpatient cases is 15% ; the mortality rate is 12% in inpatient cases and 40% in intensive care patients.3,4 d - dimer ( dd ) is a metabolic substance produced during the catabolization of fibrin by plasmin . dd levels have been shown to increase in patients who have disorders that trigger fibrin production and catabolization ; these disorders include pulmonary emboli ( pe ) , deep vein thrombosis ( dvt ) , solid tumors , leukemia , severe infections , trauma or a post - operative state , disseminated intravascular coagulation ( dic ) , pregnancy , acute stroke , sickle - cell anemia , congestive heart failure and chronic kidney failure.58 while the measurement of plasma dd levels is a well - known test for venous thromboemboli , the relationship between plasma dd and other diseases is still unclear . a limited number of studies have examined the relationship between cap and plasma dd levels . some of these studies suggest that an increase in dd is directly related to the intra- and extra - vascular coagulation that occurs in acute and chronic lung damage in cap cases.9 the present study investigated the relationship between the severity of cap and serum dd levels , as well as the potential correlation among plasma dd levels , the radiological extent of the disease and hospital mortality rates . the ultimate goal of this study was to determine whether plasma dd levels can be used to develop prognoses in cap cases . this study began in october 2007june 2008 at the department of chest diseases , faculty of medicine , cumhuriyet university , sivas , turkey , and included 60 consecutive cap patients and 24 healthy controls . the study was approved by the ethics committee of the faculty of medicine , cumhuriyet university , and all participants gave their informed consent . individuals were excluded from the study because of cancer , leukemia , known blood - clotting and bleeding disorders , disseminated intravascular coagulation ( dic ) , renal failure , rheumatological diseases , vasculitis or sickle - cell anemia , pregnancy , an age less than 18 years or thromboembolic diseases . the demographic characteristics , physical examination findings and laboratory findings ( leucocytes , hemoglobin , hematocrit , glucose , sodium , potassium , urea , creatinine , crp and sedimentation ) of all study participants were monitored regularly . initial lung x - rays were taken for all patients in the cap group and the control group . patients were placed into two groups according to their lung x - rays : lobar ( i.e. , more than one segment of the same lobe involved ) and multilobar ( i.e. , more than one lobe involved ) . patients were divided into five groups according to the pneumonia severity index ( psi ) ( psi groups i , ii , iii , iv and v).10,11 cap guidelines were used to determine whether patients would be treated at home or in the hospital.12 the criteria are shown in table 1 . the plasma dd levels of all participants were measured in the hospital s hematology laboratory before starting the treatments of their cap . blood samples taken from the antecubital vein with an injector were placed into 1.8 ml citrated tubes . the blood samples were centrifuged at 40,000 rpm for 15 minutes at room temperature and then evaluated using the quantitative latex method ( acltop , italy ) . kruskal - wallis ( cv ) , tukey s and mann - whitney u tests were used in the statistical analyses , and p - values of less than 0.05 were accepted as significant . the study included 60 cap patients with an overall mean age of 62.5 11.7 years ( 29 males with an average age of 63.0 11.7 years and 31 females with an average age of 62.0 11.7 years ) and 24 healthy controls with a mean age of 59.6 6.6 years ( 10 males with an average age of 59.5 4.6 years and 14 females with an average age of 59.4.0 7.9 years ) . the age and sex distributions for the cap and control groups are shown in table 2 . the relationship between plasma dd levels and psi statistically significant differences ( p<0.05 ) were found between the following groups : the control group and psi groups iii , iv and v ; psi group i and psi group iv and v ; psi group iii and psi group v as well as between psi group iv and psi group v. differences between the other groups were not found to be statistically significant ( p>0.05 ) ( table 3 ) . statistically significant ( p<0.05 ) differences were found between the following groups : the control group and psi group iii , iv and v ; psi group i and psi group iv and v ; psi group ii and psi group iv and v ; psi group iii and psi group iv as well as between psi group iv and psi group v. no statistically significant differences were found among the other classes ( p>0.05 ) . plasma dd levels were 337 195.1ng / ml in the outpatient group , 691.0 180.5ngml in the inpatient group , 1363.2 331.5ng / ml in the intensive care group and 161.3 38.1ng / ml in the control group ( see table 4 and figure 1 ) . in addition to cap , 38 patients had other accompanying diseases ( 15 patients : asthma / copd ( chronic obstructive pulmonary disease ) 9 patients : diabetes mellitus ; 8 patients : coronary artery disease ; 3 patients : chronic liver disease and 3 patients : peptic ulcus ) ; 22 patients had no accompanying disease . the mean plasma dd level for patients who had an accompanying disease was 776.15 473.58 , while the mean serum dd level for patients without an accompanying disease was 494.23 280.18 ng / ml . patients were divided into two groups ( lobar and multilobar ) , and plasma dd levels in the multilobar group were significantly higher than those in the lobar group ( table 5 ) ( p<0.001 ) five of the 36 inpatients ( 13.8% ) died ; all of the deceased patients had plasma dd levels that were higher than the normal limit ( 1429.8 511.3ng / ml ) , and all had another disease in addition to cap . an increase in plasma dd levels may result from the activation of the fibrinolytic system and from fibrin catabolization within the alveoli . in addition , plasma dd levels may be increased by the activation of the blood coagulation process ; this activation is caused by endotoxins in the gram - negative pathogens that trigger cap.1215 this study found that the plasma dd levels of cap outpatients , inpatients and intensive care patients correlated positively with the severity of their pneumonia . this study found a statistically significant relationship between patients psi scores and their plasma dd levels . shilon et al.,16 in a study performed on 68 cap patients and no control group , found that psi groups iv and v were associated with increased plasma dd levels when compared with groups i , ii and iii . ng / ml in the severe pneumonia group , 912.6 512.6 ng / ml in the non - severe pneumonia group and 387.94 99.56 ng / ml in the control group ; their results were statistically significant.17 ribelles et al.18 found that increased plasma dd levels were correlated with psi ; their results were also statistically significant . chalmers and coworkers19 also found that increased dd levels were correlated with psi . in their study , median dd levels were as follows : class i : 543 ng / ml ; class ii : 727 ng / ml ; class iii : 1192 ng / ml ; class iv : 1128 ng / ml and class v : 1801 ng / ml ; p<0.001 . the present study also found a statistically significant relationship between serum dd level and psi score ; these findings are consistent with the findings of previous studies . plasma dd levels were found to be statistically significant in the inpatient group than outpatients group ( defined according to the cap guidelines).12 guneysel et al . and ribelles et al . found increased plasma dd levels in intensive care patients,17,18 and shillon et al . found increased plasma dd levels in inpatients.16 the present study also found that plasma dd levels were significantly increased in intensive care patients . several studies have examined the relationship between plasma dd levels and the extent of disease in the lungs of cap patients.18,20 levi et al.20 reported a correlation between the extent of pulmonary disease , radiological appearance and plasma dd levels in severe pneumonia patients . ribelles et al.18 suggested that plasma dd levels were higher in patients with lobar or multilobar pneumonia than in patients with segmental pneumonia . no relationship was detected between a high plasma dd level and either the radiological extent of the disease , microorganisms present or the antibiotics previously administered no segmental involvement was determined in the present study . this could be because patients came to our hospital only in the later stages of the illness or because they had received treatment previously in other healthcare settings . patients were divided into two groups ( lobar and multilobar ) , and plasma dd levels in the multilobar group were significantly higher than those in the lobar group ( p<0.05 ) . ribelles et al . found a strong correlation between the mortality rate and serum dd levels in cap patients18 . while the mean serum dd level was 3,786 2,646 ng / ml in patients who died , it was only 1,609 1,808 ng / ml in patients who survived ( p < 0.0001).18 they also determined that cap patients in psi classes iv and v who had plasma dd levels higher than 2000 ng / ml had a higher risk of mortality.18 some researchers have suggested that increased plasma dd levels are related to the extent of pneumonia because the psi and apache ii score are closely associated with the mortality rate.21 low plasma dd levels in cap patients ( less than 500 ng / ml ) at the time of admission to the hospital has been linked to a decreased likelihood of an early death or major morbidity.19 several researchers have identified meaningful relationships between plasma dd levels and psi.16,17 however , because these studies included an insufficient number of patients , no strong relationship between plasma dd levels and mortality could be identified.16,17 the present study was able to identify a strong relationship between plasma dd levels and the rate of mortality ( 13.8% ) . cap patients show increased plasma dd levels even in the absence of an accompanying disease that can increase dd levels . increased plasma dd levels in cap patients were correlated with the severity of the disease , the radiological extent of the disease and in - hospital mortality rates . additional studies with more patients are needed to determine whether plasma dd levels can be used to develop prognoses for cases of pneumonia . bacterial multicomponent monooxygenases ( bmms ) comprise a family of enzymes capable of hydroxylating or epoxidizing a wide range of hydrocarbons , including the greenhouse gas methane and environmentally hazardous substances such as benzene and trichloroethylene . bmm enzymes can be grouped into four classes : the three - component enzymes soluble methane monooxygenases ( smmos ) , phenol hydroxylases ( phs ) , and alkene monooxygenases ( amos ) , and the four - component enzymes alkene / arene monooxygenases . all bmm enzymes contain three common components : a hydroxylase , a reductase , and a regulatory protein . the hydroxylase component is a multi - subunit dimeric ( 222 or 22 ) protein hosting a diiron center in each -subunit . the carboxylate - bridged diiron center is the locus for o2 activation and subsequent substrate hydroxylation / epoxidation . it is similar to those in the r2 subunit of ribonucleotide reductase , ferritin , stearoyl acyl carrier protein 9 desaturase , and the aging - related protein clk1 . the reductase component is an nadh oxidoreductase with an [ fe2s2 ] cluster in the ferredoxin domain ( fd ) and a flavin adenine dinucleotide ( fad ) cofactor in the fad domain , responsible for the reduction of the hydroxylase diiron center . the third component , a cofactor - less regulatory protein , couples nadh consumption to product formation . a key question regarding the catalytic mechanism of bmm enzymes is how component protein interactions achieve the timely control of electron transfer to the diiron active site , dioxygen activation , and hydrocarbon substrate oxidation . elucidating component protein binding sites on the hydroxylase is required as a foundation for answering such a question . an important clue comes from early crystallographic investigations of hydroxylases from the three - component system smmo and the four - component toluene / o - xylene monooxygenase ( tomo ) . both hydroxylases contain a structure of c2 symmetry with a shallow depression , termed the canyon the canyon region was proposed as the docking site for the other component proteins , and , indeed , later crystallographic studies revealed that the regulatory component does occupy a portion of the canyon in hydroxylase regulatory protein complexes of ph , toluene-4-monooxygenase ( t4mo , another four - component bmm ) , and , very recently , smmo ( figure 1b ) . regulatory protein complex of smmo ( pdb i d 4gam ) : ( a ) the hydroxylase mmoh showing the canyon , and ( b ) mmoh in complex with the regulatory protein mmob . there is another mmob molecule binding to the canyon on the other side of mmoh . mmoh -subunit is colored in green , -subunit in blue , -subunit in yellow , and mmob in purple . there is no crystal structure available for the hydroxylase reductase complex of any bmm enzyme . by using the zero - length cross - linker 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide ( edc ) , a chemical cross - linking study of smmo isolated from methylosinus trichosporium ob3b revealed that the reductase , mmor , cross - linked to the -subunit of the hydroxylase mmoh , and that the regulatory component , mmob , cross - linked to the -subunit . a different result was obtained , however , using smmo isolated from methylococcus capsulatus ( bath ) , where either the full - length mmor or its fd cross - linked to the -subunit using the same cross - linker , edc . further attempts to determine the binding site by identifying cross - linked residues failed . the two identified fd cross - linking sites , glu-56 and glu-91 , cross - linked to the n - terminal amino group of mmoh -subunit , which is not observed in the crystal structure of mmoh owing to disorder . because the mmor binding site on mmoh is obscure , it was unclear how the regulatory protein and the reductase might interact in the complete enzyme system . simulations of steady - state oxidase and oxygenase activities of smmo as a function of component protein concentrations favored a non - competitive model , whereby mmor and mmob bind at distinct sites on mmoh , forming a hypothetical ternary complex . the formation of such a ternary complex was also proposed in a small - angle x - ray scattering ( saxs ) study , where the species formed in the presence of large excess of mmob and mmor ( 1020 equiv of each relative to mmoh ) was modeled as a mmoh2mmob2mmor complex . later crystallographic investigations of the hydroxylase regulatory protein complexes , however , suggested that the regulatory component may block the reductase binding site , but there was no direct experimental evidence for such . the role of the regulatory protein in electron transfer from the reductase to the hydroxylase diiron center is also not well understood . a determination of the reductase binding site on the hydroxylase would clarify many of these questions . accordingly , in this study we determined the reductase binding site on the hydroxylase of smmo isolated from methylococcus capsulatus ( bath ) , by using hydrogen the results clearly reveal that the fd of mmor indeed binds to the canyon of mmoh . more importantly , the fd shares the same binding site as the core of mmob ; it therefore binds competitively with mmob to mmoh . consistent with the shared binding site , we show that mmob does not facilitate , but actually inhibits , electron transfer . overall , this work presents the first experimentally determined reductase fd binding site on the hydroxylase of a bmm enzyme , and it reveals how the regulatory component may control electron transfer in the catalytic cycle . 5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid ( iaedans ) was obtained from molecular probes . other chemicals were purchased from sigma - aldrich and used without further purification . the expression system for mmor ferredoxin domain ( fd , residues 1107 ) was prepared by mutating s108 and f109 of wild - type mmor to stop codons by site - directed mutagenesis , using primers shown in table s1 . fd was expressed and purified as described previously , except that an additional step with a monoq column was employed to separate apo protein without the iron sulfur cluster from the holo protein . the expression system for the mmob 2 - 33 d36c mutant was prepared by site - directed mutagenesis using mmob d36c as the template ; the primers are shown in table s1 . the mmob 2 - 33 d36c protein was expressed and purified following the procedure described for wild - type mmob . a 60 pmol portion of mmoh was incubated with fd for a final mmoh : fd concentration ratio of 1:6 during deuterium labeling . under this condition , > 95% of the fd binding sites on mmoh were saturated , based on a kd value of 0.9 m . all mixtures were incubated for 20 min at room temperature before deuterium labeling . as a control , mmoh alone was incubated in 50 mm phosphate buffer ( ph 7.0 ) and treated exactly the same as the fd - bound protein . deuterium exchange was initiated by dilution of each sample with 15-fold 50 mm phosphate buffer ( pd 7.0 ) , 99.9% d2o at room temperature . at each deuterium exchange time point ( 10 s , 1 min , 10 min , 60 min , 4 h , 6 h , and 8 h ) , an aliquot from the exchange reaction was removed and quenched by adjusting the ph to 2.5 with an equal volume of quench buffer ( 150 mm potassium phosphate buffer , h2o ) . quenched samples were immediately frozen on dry ice and stored at 80 c until analysis . several undeuterated control samples were prepared in the same way as the deuterium - labeled samples and were used for validation of the peptic peptides of the proteins used in the deuterium labeling experiments . each flash - frozen sample was rapidly thawed and injected into a waters nanoacquity with hdx technology ( waters corp . ) . the protein samples were digested online using a 2.1 mm 30 mm poroszyme immobilized pepsin cartridge ( applied biosystems ) . the digestion temperature was set to 15 c and the digestion was performed for 30 s. the cooling chamber of the ultra - performance liquid chromatography ( uplc ) system , which housed all the chromatographic elements , was held at 0.0 0.1 c for the entire time of the measurements . the injected peptides were trapped and desalted for 3 min at 100 l / min and then separated in 14 min by a 5% to 40% acetonitrile : water gradient at 40 l / min . the separation column was a 1.0 100.0 mm acquity uplc c18 bridged ethyl hybrid particles ( beh ) column ( waters corp . ) containing 1.7 m particles , and the back pressure averaged 8800 psi at 0.1 c . the average amount of back - exchange using this experimental setup was 1825% , based on analysis of highly deuterated peptide standards . deuterium levels were not corrected for back - exchange and are therefore reported as relative ; however , all comparison experiments were done under identical experimental conditions , thus negating the need for a back exchange correction . the uplc step was performed with protonated solvents , thereby allowing deuterium to be replaced with hydrogen from side chains and the amino / carboxyl terminus that exchange much more rapidly than amide linkages . the average error in determining the deuterium levels was 0.1 da in this experimental setup , consistent with previously obtained values . in order to eliminate peptide carryover , a wash solution of 1.5 m guanidine hydrochloride , 0.8% formic acid , and 4% acetonitrile was injected after each run . mass spectra were obtained with a waters xevo g2 tof instrument equipped with standard electrospray ionization source ( waters corp . ) . the instrument configuration was the following : capillary was 3.2 kv , trap collision energy at 6 v , sampling cone at 35 v , source temperature of 80 c , and desolvation temperature of 175 c . mass accuracy was ensured by calibration with 500 fmol/l human [ glu1]-fibrinopeptide b and was less than 10 ppm throughout all experiments . the mass spectra were processed with the software dynamx 2.0 ( waters corp . ) by centroiding an isotopic distribution corresponding to the + 2 , + 3 , or + 4 charge state of each peptide . deuteration levels were calculated by subtracting the centroid of the isotopic distribution for peptide ions of undeuterated protein from the centroid of the isotopic distribution for peptide ions from the deuterium - labeled sample . the resulting relative deuterium levels were automatically plotted versus the exchange - in time . identification of the peptic fragments was accomplished through a combination of exact mass analysis and ms using identity software ( waters corp . ) . ms was performed by a series of low high collision energies ramping from 5 to 32 v , therefore ensuring proper fragmentation of all the peptic peptides eluting from the lc system . iaedans - labeled mmob d36c and 2 - 33 d36c mutants were prepared following procedures described previously . concentrations of the labeled proteins were determined by using the bradford assay ( bio - rad ) . the excitation wavelength was set to 336 nm and emission was monitored at 490 nm . samples were made in 25 mm mops , ph 7.0 buffer ; the concentration of fluorescently labeled protein was 1 m . mmoh was considered to have two non - interacting binding sites ( hsite ) . the simulation procedures are described as follows , taking the titration of fd into 1 m mmoh and 1 m iaedans - labeled mmob as an example . two equilibria were considered , eqs 1 and 2 , where [ hsite ] , [ b ] , and [ fd ] are the concentrations of free mmoh binding site , free mmob , and free fd ; [ hsite - b ] and [ hsite - fd ] are the concentrations of bound mmob and fd ; [ hsite]total , [ b]total , and [ fd]total are the total concentrations of mmoh binding site ( 2 m ) , mmob ( 1 m ) , and fd ( the total amount titrated in ) . b ) was determined previously to be 0.55 m ; several kd values for the h fd complex ( kd , h fd ) were tested to allow us to choose the one that best simulated the experimental data . the concentrations [ b ] and [ hsite b ] were first calculated for each titration point by numerically solving the simultaneous eqs 1 and 2 . these values were then used to calculate the observed fluorescence anisotropy robs ( eq 3 ) , which is the sum of fluorescence anisotropy of free and bound mmob weighted by their fractional fluorescence intensity,3 where rb and rhsite b are the fluorescence anisotropy of free and bound mmob , and fb and fhsite b are the fractional fluorescence intensity of free and bound mmob , respectively . the fb and fhsite b parameters can be expressed in terms of [ b ] , [ hsite b ] , and the molar fluorescence intensity of free and bound mmob , fb and fhsite b:45 substituting eqs 4 and 5 into eq 3 , the fluorescence anisotropy of each titration point can be calculated on the basis of [ b ] and [ hsite b ] by solving eqs 1 and 2:6 the same simulation procedures were followed for the titration of fd into 1 m mmoh and 1 m iaedans - labeled mmob 2 - 33 . the kd value of 2.67 m for the h b 2 - 33 complex determined in this study was used for the simulation ( figure s3 ) . several kd values ( 0.9 , 2 , and 6 m ) for the h fd complex ( kd , h fd ) were tested to see which one best fits the experimental data . to a mixture of 10 m mmoh , 20 m mmor , and 0120 m mmob in 50 mm mops buffer , ph 7.0 , was added 10 mm cross - linker edc . the reaction was incubated at room temperature for 10 min and then quenched by adding an equal volume of sds loading buffer . the reaction was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) . the electron transfer kinetics of smmo were studied by stopped - flow optical spectroscopy at 15 c . the tubing and syringes of the hi - tech scientific sf-61 dx2 double - mixing stopped - flow instrument were made anaerobic by first flushing and then incubating with 15 mm anaerobic sodium dithionite solution for 3 h , followed by flushing with 25 mm anaerobic mops buffer , ph 7.0 right before use . the following steps were performed inside of a glovebox with an o2 level of less than 0.5 ppm . to investigate electron transfer from chemically reduced reductase , 40 m degassed fd or mmor was titrated with 3 mm sodium dithionite until the absorption at 405 nm no longer changed , and the resulting sample was then loaded into a gastight syringe . a 20 m quantity of mmoh , or 20 m mmoh premixed with 40 m mmob in the presence or absence of 10 m mmor , was degassed and sealed in another gastight syringe . to study electron transfer from nadh , a 40 m solution of anaerobic nadh was sealed in one syringe ; 20 m mmoh premixed with 40 m mmor in the presence / absence of 40 m mmob was sealed in another syringe . the syringes were then taken out from the glovebox and connected to the stopped - flow instrument . equal volumes of reagents from each syringe were rapidly mixed by the stopped - flow instrument , and the electron transfer kinetics were monitored by recording the absorbance change at 470 or 458 nm . data were fit by two ( when chemically reduced fd or mmor was used as the electron source ) or three ( when nadh was used as the electron source ) exponentials , and effective electron transfer rates were calculated as weighted averages of individual electron transfer rate constant . hdx - ms is a powerful tool for probing protein structure , dynamics , and the binding interface . the rationale behind hdx - ms relies on protein backbone amide protons that are in constant exchange with solvent protons , or deuteriums if in deuterated solvent . the number of exchangeable protons and their rates of exchange depend on factors such as ph , temperature , chemical environment , and the three - dimensional protein architecture , thus reflecting the structure and dynamics of the protein . typically , protein backbone amide protons exchange rapidly with deuterons if they are involved in weak or suboptimal hydrogen bonds , reside at / near the surface , or are readily accessible to the solvent ; the exchange rates are slower if they are involved in strong intramolecular hydrogen bonds and/or are less accessible to solvent . protein binding sites , based on the reduced solvent exposure in regions that constitute the binding interface . here mmoh is a 251.3 kda homodimer that consists of three protomer subunits in each monomer : ( 60.6 kda ) , ( 45.1 kda ) , and ( 19.8 kda ) . upon pepsin digestion , 165 overlapping mmoh peptic peptides were detected , covering 93.9% , 93.5% , and 96.4% of the sequences of the - , - , and -subunits , respectively ( figure s1 ) . the deuterium incorporation and protein dynamics were followed from 10 s up to 8 h. all the peptic peptides that were followed by hdx - ms are displayed in figure s2 . most of these peptides showed low deuterium uptake even after an 8 h incubation in deuterated buffer ( figure 2 ) . the data indicate very slow dynamics in most of the three subunits of the homodimer , suggesting that the protein is very rigid and not undergoing breathing movements indicative of a dynamic and solvent exposed structure . amide backbone hydrogens involved in hydrogen - bonding interactions in secondary structural elements such as -helices and -sheets exhibit slow exchange rates . therefore , the low deuterium uptake is consistent with the high helical content of mmoh . the and -subunits are more protected from exchange compared with the -subunit , the peptides of which seem to indicate a more accessible and dynamic structure ( figure 2 ) . summary of hdx - ms data for free mmoh in solution at four time points . the hdx - ms data are mapped onto pdb entry 1mty , with the color code indicated for deuteration times shown at the bottom of each image . the hdx - ms data are shown only on one monomer ; the second monomer is represented in sand color . we next used hdx - ms to locate regions of mmoh that show differences in exchange upon binding to the ferredoxin domain ( fd , residues 1107 , 11.8 kda ) of mmor . the two isolated domain proteins both have stable structures and retain the biochemical properties of the two domains in full - length mmor . electron transfer to the diiron(iii ) centers in the hydroxylase involves first , nadh reductions of the oxidized fad cofactor to its hydroquinone form , followed by the [ fe2s2 ] cluster in the fd sequentially shuttling two electrons from the reduced fad cofactor to the diiron center in mmoh . the fd protein is a smaller yet competent model of mmor for studying electron transfer as well as binding to mmoh . among the 165 detected peptic peptides of mmoh in the presence of fd ( figure s2 ) , six peptides derived from the -subunit and three from the -subunit exhibited lower deuterium uptake compared with samples without fd ( figures 3 and s2 ) , covering residues 7088 and 236255 of the -subunit , and residues 3748 of the -subunit . the differences in deuterium uptake were visible after 1060 min deuteration but not at earlier time points , possibly because the exchange rates in the absence of fd were already very slow due to stable h - bonds in the -helices . in the presence of fd the reduced solvent exposure at the binding site modestly reduced the exchange rates . the differences in deuterium uptake in the presence and absence of fd were subtle even at longer incubation times ( up to 8 h exchange ) , but were consistent in the set of overlapping peptides . typically , differences in deuteration greater than 0.4 da but less than 1 da are considered subtle . the experiments were performed in triplicate , and the average error of the measurements was 0.1 da . all other mmoh peptic peptides exhibited the same deuterium uptake in the presence or absence of fd ( figure s2 ) . deuterium exchange kinetics for four representative peptides that showed different deuterium uptake in the presence ( blue traces ) or absence ( red traces ) of fd : ( a ) residues 7081 , -subunit ; ( b ) residues 8288 , -subunit ; ( c ) residues 237242 , -subunit ; and ( d ) residues 4047 , -subunit . the largest number on the y - axis represents the maximum amount of deuterium that can be incorporated in each peptic peptide . we then mapped the peptides that showed decreased deuterium uptake in the presence of fd onto the crystal structure of mmoh ( pdb entry 4gam ) . these peptides cluster in the canyon region at the 22 interface , representing a possible fd binding site ( figure 4a ) . this region includes the area closest to the diiron center from the protein surface , a preferred binding site for the iron sulfur cluster of the reductase in order to facilitate fast electron transfer . this binding site determined by hdx - ms rationalizes the seemingly conflicting results of previous chemical cross - linking studies . because the binding site consists of residues from both - and -subunits , mmor can cross - link to either the -subunit ( for smmo isolated from methylococcus capsulatus ( bath ) ) or the -subunit ( for smmo isolated from methylosinus trichosporium ob3b ) . it is unlikely that smmos isolated from different species have different reductase binding sites . the different cross - linking results are most likely due to different distributions of carboxyl groups and amino groups that are required to be in close proximity for the cross - linking mediated by edc . ( a ) mmoh peptides that showed decreased deuterium uptake in the presence of fd are mapped onto the crystal structure of mmoh ( pdb i d 4gam ) . ( c ) a closer view of the binding interface in the docked model : the [ fe2s2 ] cluster of fd , mmoh residues at the binding interface , and the diiron center of mmoh , viewed from the top of the figure . ( d ) the crystal structure of mmoh-2mmob , showing that mmob covers the fd binding site . mmoh is colored in cyan ; binding site peptides of the -subunit in orange and those of the -subunit in yellow ; fd in red ; and mmob in purple . to validate the hdx - ms - determined fd binding site , we performed computational docking using the cluspro server . the structure of fd ( residues 198 , pdb i d 1j4q ) previously determined by solution - state nmr spectroscopy and the crystal structure of mmoh ( pdb i d 4gam ) were used as input structures , and the nmr - determined fd residues involved in binding to mmoh were used as docking constraints . the results show that fd covers the hdx - ms - determined binding surface ( figure 4b ) . the -subunit peptides involved in binding ( yellow region in figure 4 ) seem uncovered in the docked structure ( figure 4b ) , but upon a closer examination residues 4548 inside the canyon were found to be in close contact with fd . the docking result is therefore in full agreement with the hdx - ms data . a close examination of the docked mmoh fd complex reveals that the diiron active site of mmoh is approximately 14 away from the [ fe2s2 ] cluster of fd , a favorable distance for electron transfer . consistent with hdx - ms results , the docked model reveals that fd covers the pore region of mmoh , which we previously proposed as the proton transfer pathway from the mmoh surface to its diiron center . residue e240 in the mmoh -subunit , the gating residue of the pore , is situated midway between the [ fe2s2 ] cluster of fd and the diiron center of mmoh ( figure 4c ) . previously , this residue was found to shift its conformation toward the protein interior upon mmob binding , closing the pore and possibly bringing in a proton to the diiron active site . we propose that an identical conformational change occurs when mmor binds to mmoh during electron transfer from the fd [ fe2s2 ] cluster to the carboxylate - bridged diiron center , providing the mechanism for proton - coupled electron transfer . strikingly , the fd binding site determined here overlaps largely with that of mmob as previously determined by the crystal structure of the mmoh2mmob complex . in particular , this structure reveals that the core of mmob docks into the canyon region , covering the very same area where we now conclude fd binds , while the n - terminal tail of mmob binds to an adjacent location on mmoh , on the surface of helices h and 4 of the -subunit , adopting a ring - like conformation ( figure 4d ) . this observation requires the fd of mmor and the core of mmob compete for the same binding site in the canyon of mmoh . to test this possibility , we first investigated the binding competition between fd and the core of mmob by fluorescence anisotropy titrations . the mmob core was prepared by truncating the n - terminal tail at residue 33 ( 2 - 33 ) , and a cysteine mutation d36c was introduced in order to attach the fluorophore iaedans . the n - terminal truncated mmob ( designated mmob 2 - 33 ) is still able to bind to mmoh but with lower affinity ( kd = 2.67 m , figure s3 ; kd = 0.55 m for the full - length mmob ) . to characterize the binding competition , fd was titrated into a mixture of 1 m mmoh and 1 m iadeans - labeled mmob 2 - 33 . the fluorescence anisotropy decreased steadily as increasing amounts of fd were added ( figure 5a ) , indicating displacement of n - terminal truncated mmob by fd from mmoh ( scheme 1a ) . the titration curve could be simulated by assuming that the n - terminal truncated mmob and fd compete for the same binding sites on mmoh ( mmohsite , one on each side of mmoh ) , and an apparent kd value of 2 m was obtained for the mmoh fd complex on the basis of the simulations ( figure 5a ) . experimental evidence for mmor and mmob binding competition . ( a , b ) titrating fd into 1 m mmoh and 1 m iadeans - labeled n - terminal truncated ( 233 ) mmob ( a ) or full - length mmob ( b ) . the titration curves are simulated assuming kd = 0.9 , 2 , and 6 m for the mmoh fd complex ; kd = 2 and 6 m best fit the experimental data in ( a ) and ( b ) , respectively . edc was used as the cross - linking reagent , and 0 , 1 , 2 , 4 , and 6 equiv mmob relative to mmor were added to a mixture of 10 m mmoh and 20 m mmor . the cross - linking reaction mixtures were resolved by sds - page , and bands corresponding to the mmor-subunit cross - linking product are shown . in the binding competition between fd and full - length mmob ( b ) , fd may displace the core of mmob from the canyon , but the n - terminal tail may still bind to mmoh . mmoh complex when the titration experiment was repeated with the full - length mmob labeled with iaedans ( figure 5b ) , indicating that fd is less effective in displacing full - length mmob . this result is an expected consequence of the additional binding site of the n - terminal tail of mmob on the surface of helices h and 4 of the mmoh -subunit , a binding region not shared with fd . it is possible that the n - terminal tail serves as an anchor attached to the surface of mmoh , such that , when fd displaces the core of mmob from the canyon , mmob does not completely dissociate from mmoh ( scheme 1b ) . a simple competitive binding model would therefore no longer apply to the component interactions in this case . similar results were obtained by using the full - length mmor as the titrant . an apparent kd value of 8 m for the mmoh mmor complex is required to fit the titration curve using a competitive binding model ( figure s4 ) , considerably larger than the 0.9 m kd value previously determined for the mmoh mmor complex in the absence of mmob . these results clearly demonstrate that both fd and the full - length mmor are able to displace mmob , in particular the core of mmob , from mmoh . this property was demonstrated by studying the effect of mmob on mmor cross - linking to mmoh . as shown in figure 5c , mmor forms cross - links to the mmoh -subunit in the presence of edc , as demonstrated previously ; the yield of this cross - link decreases in a dose - dependent manner as the mmob concentration increases from 0 to 6 equiv relative to the mmor concentration , indicating that mmob blocks mmor binding , and thus its cross - linking , to mmoh . a similar finding the determination of component protein binding sites on the hydroxylase and their binding competition form a basis for understanding the role of the regulatory protein in electron transfer reactions of smmo . previously , mmob was proposed to facilitate electron transfer from mmor to mmoh , and this effect was ascribed to formation of a hypothetical mmoh2mmob2mmor ternary complex . it was hypothesized that the increased electron transfer exhibited by this ternary complex is due to a conformational change of mmoh induced by mmor . considering the substoichiometric concentration of mmor found in vivo and used during activity assays , it was further proposed that such conformational changes are retained throughout the catalytic cycle even after mmor dissociates from the ternary complex . considering our current finding that the core of mmob and the fd of mmor compete for binding to the canyon of mmoh , these proposals are deemed unlikely . because mmob can inhibit mmor binding to the canyon , mmob would inhibit electron transfer as well . to test experimentally whether mmob increases the electron transfer rate , and whether pre - equilibrating mmor with other protein components would result in such an increase , we designed two sets of single - turnover , single - mixing stopped - flow experiments . in the first set , the electron transfer reactions were initiated by mixing 2 equiv of chemically reduced fd to ( i ) 1 equiv of oxidized mmoh alone , ( ii ) 1 equiv of oxidized mmoh equilibrated with 2 equiv of mmob , and ( iii ) 1 equiv of oxidized mmoh equilibrated with 2 equiv of mmob as well as 0.5 equiv of mmor ( figure 6a ) . the reactions were monitored at 470 nm , which increases in intensity as electrons are transferred to the mmoh diiron sites from the reduced [ fe2s2 ] clusters . the previous proposal would predict that case ( iii ) , where all three components were pre - mixed and equilibrated , would exhibit the fastest electron transfer rate . the actual results ( figure 6b ) , however , showed that reaction ( i ) displays the fastest electron transfer rate . including mmob in reactions ( ii ) and ( iii ) significantly retarded the electron transfer reaction , even when mmor was added and pre - equilibrated with other components . these experiments were repeated using chemically reduced full - length mmor as the reductant , and the same trend was observed ( figure s5 ) . mmob inhibits electron transfer , using chemically reduced fd as the electron source . the electron transfer reaction was initiated by mixing 2 equiv of nadh with ( i ) 1 equiv of oxidized mmoh and 2 equiv of mmor and ( ii ) 1 equiv of oxidized mmoh , 2 equiv of mmob , and 2 equiv of mmor ( figure 7a ) . the reactions were monitored at 470 nm , where the absorption first decreased due to the reduction of the fad cofactor by nadh and the subsequent intra - mmor electron transfer to the [ fe2s2 ] cluster . the absorption at this wavelength then increased , as electrons were transferred from these cofactors to the diiron centers in mmoh . the previous proposal would predict that reaction ( ii ) , which had all three protein components pre - equilibrated , would display the faster electron transfer rate , but in fact it is slower ( figure 7b ) , again demonstrating the inhibitory effect of mmob on electron transfer ( table 1 ) . electron transfer reactions were initiated by mixing the reagent in syringe 1 with protein(s ) in syringe 2 in single - mixing stopped - flow experiments ( setups also shown in figures 6a and 7a ) . the inhibitory effect of mmob on electron transfer is in full agreement with our finding that the core of mmob shares a binding site on mmoh with the fd of mmor such that mmob inhibits mmor binding . although electron transfer is retarded , it is still much faster than the rate of substrate turnover ( kcat = 0.1 s at 15 c using propylene as substrate ) . the advantage of such competitive binding is that it provides a mechanism for modulating electron transfer during catalysis . by fine - tuning the affinity of mmor and mmob for the canyon region of mmoh , the binding of mmor can be inhibited during dioxygen activation , preventing undesired electron transfer that could quench activated dioxygen intermediate species . such quenching is suggested by oxidase activity displayed by mmoh and mmor in the absence of mmob . the modulation of electron transfer exhibited by the mmob regulatory protein is most likely a feature common to other bmm enzymes . these enzymes share conserved protein sequences as well as structures , and , like smmo , their regulatory proteins also bind to an analogous canyon region of the hydroxylase , as illustrated in the x - ray structures of the protein complexes in ph and t4mo , blocking the shortest pathway to the hydroxylase diiron center from its surface . the reductase may therefore need to compete with the regulatory protein for binding in order to deliver electrons to the diiron centers in these enzymes as well . in disagreement with this hypothesis , previous studies of ph and tomo these electron transfer rates , however , were determined in steady - state assays in the absence of substrates , by measuring nadh consumption . under these conditions , the nadh consumption rate depends on the oxidase activity of the hydroxylase , and the accelerated nadh consumption in the presence of the regulatory component is most likely due to increased oxidase activity and not an indication of accelerated electron transfer . using hdx - ms , the binding site for the ferredoxin domain of mmor was determined to be in the canyon of mmoh , the same region where the core of the mmob regulatory protein binds . this finding is consistent with previous chemical cross - linking results and the current computational docking study , as well as a series of binding competition assays . mmob inhibits mmor binding to the canyon as well as the electron transfer that leads to reduction of the hydroxylase . the previous proposals that mmob increases the electron transfer rate when all three components are pre - equilibrated has been tested experimentally and proved to be invalid . regulatory proteins of other bmm enzymes may similarly share binding sites with their reductases and inhibit electron transfer . such binding competition would provide a control mechanism for electron transfer in bmm enzymes . to initiate the catalytic cycle , the reductase displaces the regulatory protein from the canyon of the hydroxylase , reducing the diiron center from diiron(iii ) to diiron(ii ) ; the regulatory protein then re - binds to the canyon and displaces the reductase , initiating o2 activation and substrate oxidation . the binding affinities of the component proteins may be fine - tuned so that the reductase is unable to displace the regulatory protein at this step , preventing quenching of activated oxygen intermediates p * , hperoxo , and q. at the end of the catalytic cycle , the diiron center returns to the diiron(iii ) state , and the reductase can bind again to the canyon , priming the enzyme for the next cycle of catalysis .

background : plasma d - dimer levels are directly related to the intra- and extra - vascular coagulation that occurs in acute and chronic lung damage in patients with community - acquired pneumonia ( cap).objectives : this study examines the relationship between the severity of community - acquired pneumonia and d - dimer levels . in addition , the study examines the correlations among community - acquired pneumonia , the radiological extent of the disease and mortality.methods:the pneumonia severity index was used to classify patients into five groups . patients were treated at home or in the hospital according to the guidelines for community - acquired pneumonia . blood samples were taken from the antecubital vein with an injector and placed into citrated tubes . after they were centrifuged , the samples were evaluated with the quantitative latex method.results:the study included 60 patients who had been diagnosed with community - acquired pneumonia ( mean age 62.5 11.7 ) and 24 healthy controls ( mean age 59.63 6.63 ) . the average plasma d - dimer levels were 337.3 195.1ng / ml in the outpatient treatment group , 691.0 180.5 in the inpatient treatment group , 1363.2 331.5 ng / mlin the intensive care treatment group and 161.3 38.1ng / ml in the control group ( p<0.001 ) . the mean d - dimer plasma level was 776.1 473.5ng / ml in patients with an accompanying disease and 494.2 280.1 ng / ml in patients without an accompanying disease ( p<0.05).conclusions : plasma d - dimer levels were increased even in community - acquired pneumonia patients who did not have an accompanying disease that would normally cause such an increase . the hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent monooxygenases ( bmms ) requires the interplay of three or four protein components . how component protein interactions control catalysis , however , is not well understood . in particular , the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s ) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic . here we determine the reductase binding site on the hydroxylase of a bmm enzyme , soluble methane monooxygenase ( smmo ) from methylococcus capsulatus ( bath ) . we present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase , previously determined to be the regulatory protein binding site as well . the latter thus inhibits reductase binding to the hydroxylase and , consequently , intermolecular electron transfer from the reductase to the hydroxylase diiron active site . the binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer , and other bmm enzymes are likely to adopt the same mechanism .

a 46-year - old married saudi woman presented at a primary health care center with left upper abdominal pain , with no other associated symptoms . five months later , she presented to the surgery clinic because of increased abdominal pain with no response to medication ; her physical examination showed fullness of the left upper abdomen . her routine laboratory investigation as well as chest and abdominal x - rays were not remarkable . ultrasound of the abdomen showed a solid mass at the left hypochondrium separated from the spleen and just below the left hemidiaphragm . computed tomography ( ct ) scan of the abdomen ( figure 1 ) showed encapsulated hypodense mass at the left upper abdomen which was interposed but clearly separated from left liver lobe , spleen , stomach and left hemidiaphragm . magnetic resonance imaging ( mri ) of the abdomen ( figure 2 ) confirmed the location of the mass and attachment to the inner aspect of the anterior abdominal wall but separated from internal abdominal organs . the mass was isosignal intensity to muscles in t1- weighted images and heterogeneous hyperintense signal in t2 weighted images . in post ( a ) axial non - enhanced ct scan of the abdomen showing a well defined hypodense mass at the left hypchondrium , interposed between left liver lobe , left hemidiaphragm and stomach . ( a ) axial t1w1 : an oval shape mass with signal intensity parallel to the muscle . ( c ) axial post contrast t1w1 showing avid contrast enhancement of the mass . at surgery , a firm , rounded mass apparently encapsulated at the left sub - diaphragmatic area was found attached to the extra - peritoneal fascia of the anterior abdominal wall . histopathology showed a biphasic tumor composed of spindle cell and epithelial cell elements , with mild anaplasia & infrequent mitosis . the final diagnosis was low grade synovial sarcoma involving the fascial aponeurosis of the anterior abdominal wall . synovial sarcoma ( ss ) is an uncommon soft - tissue malignant tumor that is common in the extremities of middle - aged patients , close to large joints particularly the knee in the popliteal fossa.12 despite its name , the lesion does not commonly arise in an intraarticular location but usually near joints . the tumor arises from pleuripotential mesenchyme , in close association with joint capsules , tendon sheaths , bursae and fascial structures . it is generally accepted that synovial sarcoma is derived from primitive mesenchymal cells , not synovial cells.2 synovial sarcoma is the fourth most common type of soft tissue sarcoma following malignant fibrous histiocytoma , liposarcoma and rhabdomyosarcoma . synovial sarcoma accounts for 5 - 10% of all soft tissue sarcomas.3 about 85 - 90% of ss occur in the extremities.4 reported examples arising in the anterior abdominal wall are rare . synovial sarcoma in the abdominal wall tends to occur with a much greater frequency in females in contrast to such tumors in the extremities or the neck which tend to occur with a much greater frequency in males.5 radiological findings of ss are not pathognomonic . however , findings of a soft - tissue mass , particularly if calcified ( 30% ) , near but not in a joint of a young patient , are very suggestive of the diagnosis . cross - sectional imaging features are essential for staging extent of the tumor and the planning of surgical resection.6 the most common ct appearance of ss is that of a heterogeneous soft - tissue mass with attenuation similar to or slightly lower than that of muscle areas of lower attenuation representing necrosis or hemorrhage are also common with heterogeneous contrast enhancement.7 on mri , ss typically appears as a prominently heterogeneous multilobulated soft - tissue mass with signal intensity similar to or slightly higher than that of muscle on t1-weighted mr images . prominent heterogeneity with predominant high signal intensity is also a feature of ss on t2-weighted mr images . the presence of multilobulation and marked heterogeneity are highly suggestive of a diagnosis of ss . ct and mri are useful in defining the extent of the disease and in follow up response to chemotherapy.7 the presence of extensive calcification suggests a more favorable prognosis.8 similar ct and mri appearances are seen in our case . despite its name there are three main histologic subtypes of ss : biphasic , monophasic , and poorly differentiated types . poorly differentiated ss is generally epithelioid in morphology and has high mitotic activity.78 in the present case , histopathology showed a biphasic pattern of ss , the coexistence of spindle cells in a wavy pattern , and the pseudoglandular formation . both biphasic and monophasic synovial sarcomas are usually intermediate grade ( grade 2/3 ) ; however , both types can be high grade ( grade 3/3 ) . poorly differentiated synovial sarcomas are high - grade tumors.7 the specific chromosomal aberration in synovial sarcoma has recently been reported . the hallmarks for synovial sarcoma are the ( x ; 18 ) translocation and syt - ssx gene fusion products . is the molecular diagnosis in synovial sarcoma , as well as immunohistochemical study , especially in cases in which histological diagnosis is difficult.79 surgical excision is the treatment of choice , and the recurrence rate range from 28% to 36% even with adequate surgical and adjunctive therapies.4 however , the multimodality treatment approach has improved the prognosis of synovial sarcomas . tumor invasiveness , histologic grade and tumor size significantly correlate with the survival period , with a more preferable prognosis of synovial sarcomas encountered in childhood . chemotherapy has been used to treat metastatic or residual disease.710 primary synovial sarcoma of the anterior abdominal wall is a rare extra - articular tumor site . in cases of anterior abdominal wall masses , docetaxel ( dcx ) is a second generation taxane , derived from the inactive 10-deacetyl baccatin iii , extracted from the european yew tree ( taxus baccata ) . dcx has better water solubility , pharmacokinetic profile , and anticancer activity than paclitaxel . current fda approved dcx products , including taxotere , are essentially tween 80/ethanol - based solutions , which unfortunately are associated with various significant side effects . hypersensitivity reactions , which are attributed to the tween 80 in the formulations , can vary from simple skin rash to systemic anaphylaxis and necessitate premedication with corticosteroids . other problems associated with the tween 80/ethanol - based dcx formulations include the nonspecific accumulation of dcx in healthy organs , which may lead to systemic toxicity and subsequent discontinuation of therapy . nanoparticle - based , tween 80-free dcx formulations are expected to not only avoid tween 80-related side effects but also increase the concentration of dcx in tumors due to the enhanced permeation and retention ( epr ) effect . data from many previous studies demonstrate that nanoparticles of 100200 nm are most successful in tumor vasculature extravasation , although there are disagreements in the literature . the heterogeneous nature of tumor type , size , location , and metastasis may contribute to the disagreements . in order to improve the epr - related nanoparticles extravasation , nanoparticles should be designed to circulate longer in the blood , while the drug of interest is retained within the nanoparticles . pegylation is a strategy to render the surface of nanoparticles hydrophilic , thus enabling the nanoparticles to evade early opsonization and circulate longer in the blood . on the other hand , for a drug to be retained within the nanoparticles , a strong affinity between the drug and the excipient(s ) used to prepare the nanoparticles is required . solid lipid nanoparticles ( slns ) have been extensively investigated as drug carriers . advantages of such nanocarriers include high compatibility with lipophilic drugs , ease of fabrication , and controlled release . reported the development of a pegylated lipid nanocapsule formulation ( lnc ) for paclitaxel using a novel phase inversion - based method . the resultant lncs were made of an oily medium - chain triglyceride core and stabilized with soybean lecithin as a lipophilic surfactant , and peg hydroxystearate ( solutol ) as a hydrophilic surfactant . applied a high pressure homogenization technique to prepare a sln formulation of paclitaxel using triglyceryl myristate ( trimyristin ) and phospholipids . the formulation showed improved in vitro activity , but the in vivo circulation time and biodistribution profile were not improved , as compared to the market product taxol . applied a factorial design to optimize formulation parameters to prepare paclitaxel sln formulations using compritol 888 ato ( a mixture of mono- , di- , and triglycerides of behenic acid ) and precirol ato5 ( i.e. , glyceryl palmito - stearate ) , and the final optimized formulation demonstrated an improved in vitro cytotoxic activity against the murine breast cancer cell line mxt - b2 . the present study aimed at the rational selection of a triglyceride from a list of medium- and long - chain triglycerides for the development of a sln formulation to ultimately improve the antitumor activity of dcx . previously it was reported that low melting point triglycerides are excellent solubilizers for dcx , prompting us to hypothesize that high melting point triglycerides will be suitable excipients for preparing dcx - incorporated slns . triglycerides that are solid at body temperature were selected to ensure formulation stability and to avoid droplet coalescence . an oil - in - water ( o / w ) emulsion - based method was applied , where dcx and all lipid components were dissolved in the oil phase , and the aqueous phase consisted of a 0.1% ( w / v ) poloxamer 188 aqueous solution . finally , the in vitro and in vivo antitumor activities of the selected formulation were evaluated . the 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-[methoxy ( polyethylene glycol)-2000 ] ( dope - peg-2000 ) and phosphatidylcholine from chicken egg ( epc ) were from avanti polar lipids , inc . sepharose 4b , mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) kit , tween 80 ( t80 ) , poloxamer 188 ( pluronic f68 ) , trimyristin ( tm ) , trilaurin ( tl ) , tristearin ( ts ) , tripalmitin ( tp ) , mannitol , sucrose , phosphate buffer saline ( pbs , ph 7.4 ) , triglyceride assay kit , and caspase 3 assay kit were all from sigma - aldrich ( st . louis , mo , usa ) . float - a - lyzer dialysis tubes ( mwco 50,000 ) were from spectrum chemicals & laboratory products ( new brunswick , nj , usa ) . tc-1 cells ( murine lung cancer cell line ) were from the american type culture collection ( atcc , rockville , md , usa ) and grown in rpmi 1640 , supplemented with 10% fetal bovine serum ( fbs ) and 1% of 100 g / ml streptomycin and 100 iu / ml penicillin ( 1% p / s ) . m - wnt cells ( murine mammary gland cell lines ) were from dr . m - wnt cells were grown in a similar medium as tc-1 , with an additional supplement of 1% glutamax . human breast adenocarcinoma cells ( mda - mb-231 ) were from atcc and grown in dmem supplemented with 5% fbs and 1% p / s . all cell culture reagents were from invitrogen ( life technologies , carlsbad , ca , usa ) . female c57bl/6 mice ( 68 weeks old ) were from charles river laboratories ( wilmington , ma , usa ) . briefly , 1 ml of dichloromethane ( dcm ) containing dcx , a triglyceride ( tm , tp , tl , or ts ) , egg pc , and dope - peg-2000 in a weight ratio of 1:20:10:2 was added to 10 ml of 0.1% poloxamer 188 aqueous solution in a glass vial , and the mixture was sonicated using a probe sonicator , with a microprobe attached , for 40 s , at a sonication intensity of 50% ( q - sonica llc , newtown , ct , usa ) . the glass vial was placed in an ice bath during sonication to prevent heat accumulation . the emulsion was stirred for 15 min at 400 rpm in a water bath ( 65 c ) to evaporate dcm , and was then stirred for an additional hour at room temperature . the resultant nanoparticle suspension was concentrated to 1 ml by ultrafiltration using an amicon device ( millipore inc . , 30,000 mwco ) ( 490 g , 25 min , 4 c ) as previously reported . finally , slns were briefly sonicated to eliminate aggregates due to the concentrating process . dcx - free slns were prepared similarly without the addition of dcx . for slns that were used in animal studies , the ultrafiltration period was extended to 60 min to further concentrate the suspension . the prolonged ultrafiltration did not result in any significant particle size change ( data not shown ) . kansas city , mo , usa ) with 9.25% ( w / v ) sucrose as a cryoprotectant . the tween 80/ethanol - based dcx formulation ( dcx in t80/e ) was prepared by dissolving dcx in tween 80 ( 20 mg / ml ) . this concentrate was then diluted with water / ethanol solution to make a final dcx solution of 4 mg / ml . the final concentrations of tween 80 and ethanol in the solution were 20% ( v / v ) and 13% ( v / v ) , respectively . particle size and zeta potential of the slns were measured using a malvern zetasizer nano zs ( malvern instruments , worcestershire , u.k . ) . briefly , 20 l of the concentrated slns in suspension were diluted to 1 ml with water , and the particle size and zeta potential were determined at room temperature . the slns were examined using an fei tecnai transmission electron microscope ( fei corporate , hillsboro , or , usa ) at the institute for cellular and molecular biology , microscopy and imaging facility at the university of texas at austin as previously reported . the content of dcx in the slns was determined using hplc after extraction as previously reported with modifications . briefly , slns in suspension were diluted 510 times with methanol in a glass vial , which was placed in a water bath ( 65 c ) for 20 min to dissolve the lipids , and placed at 20 c for 45 min . the supernatant was collected by centrifugation at 18000 g for 10 min at 4 c ( beckman coulter inc . , brea , ca , usa ) , and 5 l of the supernatant was used for hplc assay as previously described . , santa clara , ca , usa ) , with rp - c18 column ( zorbax eclipse , 5 m , 4.6 mm 150 mm ; santa clara , ca , usa ) . the mobile phase was acetonitrile and water ( 1:1 , v / v ) . the flow rate was 1 ml / min , and the detection wavelength was 230 nm . the dcx loading percentage was measured using a similar procedure , with the exception that the slns were lyophilized , and 5 ml of methanol was added to 5 mg of the lyophilized slns . the weight percentage of dcx in the slns ( % w / w ) was calculated based on the following formula : to investigate whether free dcx coexisted with dcx - slns in the nanoparticle preparation , the slns ( 100 l ) were applied to a sepharose 4b column ( 6 mm 30 cm ) equilibrated with water , and the dcx - slns were eluted with water . fractions of 0.5 ml were collected , and 0.3 ml of each fraction was lyophilized to determine the content of dcx as mentioned above . in addition , the absorbance of each fraction ( 100 l ) at 500 nm was measured using a biotek synergy ht multi - mode microplate reader ( winooski , vt , usa ) to determine their turbidity , which was used as an indication of the presence of nanoparticles in the fractions collected . finally , the concentration of triglycerides in each fraction was also measured using a sigma triglyceride assay kit following the manufacturer s instruction . dcx - slns prepared using different triglycerides were stored in parafilm - sealed vials at 4 c for eight days . particle size , zeta potential , and dcx content were measured as mentioned above shortly after the preparation and on day 8 to monitor any change of these parameters . the release of dcx from the slns made with different triglycerides was monitored using float - a - lyzer tubes ( mwco 50,000 ) . briefly , dcx - slns suspension was diluted to 1 ml with pbs and transferred to the dialysis tube , which was then placed in a 50 ml plastic tube containing 20 ml of release medium ( pbs , 0.1 mm , ph7.4 , with 1% tween 80 ) . the tubes were then placed at 37 c in an orbital shaker at 100 rpm ( max - q 5000 , thermo scientific , waltham , ma , usa ) . at predetermined time points , the whole release medium was replaced with fresh medium to maintain sink condition , and dcx concentration was analyzed using hplc as previously mentioned . the release of dcx from the dcx in t80/e formulation was evaluated similarly for comparison . for mdsc , a ta instruments model 2920 dsc ( new castle , de , usa ) was used , and the data were analyzed using ta universal analysis 2000 software . the ramp rate was 5 c / min , and the temperature range was from 10 to 200 c . samples included dcx , trimyristin , dcx - slns ( prepared with trimyristin ) , blank slns , and the physical mixture of dcx and blank slns . a philips model 1710 x - ray diffractometer ( philips electronic instruments inc . , mahwah , nj , usa ) available in the texas materials institute x - ray facility at the university of texas at austin was used to analyze the crystallinity of dcx in the slns . samples included dcx alone , dcx - slns ( prepared with trimyristin ) , dcx mixed with blank slns , and blank slns . cells were seeded in 96-well plates at a density of 3,000 cells / well and incubated at 37 c with 5% co2 overnight . they were treated with various concentrations of dcx - slns ( prepared with trimyristin ) , dcx in t80/e , blank slns , or t80/e alone for 72 h. cell viability was determined using an mtt assay as previously described . ic50 values were calculated using graphpad prism ( graphpad software , inc . , la jolla , ca , usa ) . caspase 3 activity was determined using a sigma - aldrich caspase 3 fluorimetric assay kit . in brief , tc-1 cells were seeded in 24-well plates at 25,000 cells / well and incubated overnight . the cells were treated with dcx - slns ( prepared with trimyristin ) , dcx in t80/e , blank slns , or t80/e for 72 h. the concentration of the dcx was 0.01 m . the supernatant was transferred to a clear - bottomed black plate and mixed with the assay substrate , acetyl - asp - glu - val - asp-7-amido-4-methylcourmarin ( ac - devd - amc ) . the mixture was incubated for 6 h for the hydrolysis of the ac - devd - amc by caspase 3 to release the fluorescent amc , which was quantified by measuring the fluorescence intensity at 360 nm ( excitation)/460 nm ( emission ) according to the manufacturer s instruction . the unit of the caspase 3 activity was mol amc / min / ml . a caspase 3 inhibitor ( provided in the kit ) total protein concentration in the cell lysates was determined using bio - rad dc protein assay kit following the manufacturer s instruction . all animal protocols were approved by the institutional animal care and use committee at the university of texas at austin , and the national institutes of health ( nih ) guidelines for laboratory animal use and care were followed . animals were left to acclimatize for at least 7 days upon arrival from the vendor . each mouse was injected with the murine tc-1 lung cancer cells ( 5 10 cells per mouse ) suspended in 100 l of fbs - free rpmi 1640 medium subcutaneously in the shaved left flank . six days after the implantation ( day 6 ) , mice were randomized into 4 groups , 7 mice per group , and injected intravenously via the tail vein with dcx - slns ( prepared with trimyristin ) , dcx in t80/e , blank slns , or 5% mannitol as a vehicle control . tumor sizes were measured using a digital caliper , and tumor volumes were calculated using the following formula : on day 21 , mice were euthanized to harvest tumor tissues , which were weighed , fixed in zn formalin buffer for immunohistochemistry . tissue sample preparation for immunohistochemical evaluation was carried out in the histology and tissue analysis core at dell pediatric research institute ( dpri ) at the university of texas at austin . the formalin - fixed tumor tissues were embedded in paraffin wax , sectioned , and stained with an antibody against cd-31 ( abcam , cambridge , ma , usa ) as a marker for angiogenesis ( n = 3 ) . slides were then scanned , and images were taken using the scanscope xt ( aperio technologies , vista , ca , usa ) . three weeks after tumor implantation , mice were divided into 2 groups ( n = 910 ) ; one group was injected with dcx in t80/e ( equivalent to dcx dose of 16 mg / kg ) via the tail vein , and the other group with dcx - slns prepared with trimyristin ( equivalent to dcx dose of 16 mg / kg ) . two or twelve hours later , 45 mice from each group were euthanized to collect tumor , liver , kidney , spleen , heart , lung , and blood samples . the organs and tumor tissues were weighed and then stored at 80 c the blood samples were mixed with an edta solution and allowed to stand for about 15 min and centrifuged ( 3300 g , 10 min , 4 c ) to separate the plasma , which was stored at 80 c . dcx was extracted from the samples using ethyl acetate , and dcx concentrations in the samples were determined using hplc . statistical analyses were completed by performing anova followed by fisher s protected least significant difference procedure . a p - value of 0.05 ( two - tail ) was considered significant . the nanoparticles were composed of dcx , a triglyceride , epc , dope - peg-2000 , and pluronic f68 . the average particle size of slns produced using tristearin , tripalmitin , trimyristin , and trilaurin as the triglyceride was 178.4 2.3 , 176.3 3.9 , 182.8 2.0 , and 150.7 14.5 nm , respectively ( table 1 ) . the polydispersity indices of all nanoparticle preparations were equal to , or below , 0.2 . the content of dcx in the final slns was 2.42.8% ( w / w ) ( table 1 ) . the release of dcx from the slns was slower , relative to the diffusion of dcx out of the dcx in t80/e formulation ( figure 1a ) . only about 4.59% of the dcx was released from the sln formulation within the first 6 h , whereas about 31% of dcx diffused out of the t80/e formulation within the same time period ( figure 1a ) . the rate at which the dcx was released from the dcx - slns prepared with trimyristin was the slowest ( figure 1b ) . ( a ) the release of dcx from dcx - slns ( closed circles ) prepared using tristearin ( ts ) , tripalmitin ( tp ) , trimyristin ( tm ) , or trilaurin ( tl ) . as a control , the release of dcx from dcx in t80/e ( open circles ) was also included . ( b ) a comparison of the percent of dcx released per hour from dcx - slns prepared using different triglycerides . the release rates were calculated with data in the initial 24 h period . each point represents mean ( n = 3 ) . during a short - term , 8-day stability study at 4 c , no significant change in particle size and dcx content in any of the four dcx - slns preparations was found ( figures 2a , b ) . however , the zeta potential of dcx - slns prepared with tripalmitin and trilaurin changed significantly ( figure 2c ) . based on data shown in figures 1 and 2 , the dcx - slns prepared with trimyristin were chosen for further studies , because the release of dcx from the slns prepared with trimyrstin as the triglyceride was the slowest , and the resultant dcx - slns were also relatively more stable . the particle sizes ( a ) , dcx contents ( b ) , and zeta potentials ( c ) of dcx - slns prepared with different triglycerides shortly after preparation or after 8 days of storage at 4 c . shown in figure 3a are the gpc results for the dcx - slns prepared with trimyristin as the triglyceride . about 90% of the dcx that was eluted from the column was associated with the triglyceride and with the fractions that contained the nanoparticles ( calculated based on the area under curves of the gpc profiles ) ( figure 3a ) . the slns were successfully lyophilized with 9.25% ( w / v ) sucrose as a cryoprotectant ( data not shown ) . ( a ) a representative gpc profile of dcx - slns prepared with trimyristin . collected fractions were divided into three portions : one was analyzed for dcx content ( red ) , one was used to measure turbidity ( od 500 nm ) ( blue ) , and the other one was used to measure trimyristin concentrations , and the visible absorbance of the formed colored component was measured ( od 540 ) ( black ) . ( b ) a representative tem image of dcx - slns prepared with trimyristin ( bar = 200 nm ) . dsc analysis of the dcx - slns , free dcx , blank slns , and blank slns mixed with dcx showed that dcx exhibited a characteristic melting peak at 167.4 c ( figure 4a ) . the physical mixture exhibited an endothermic melting peak at 143.4 c , which can be attributed to the presence of free dcx , as the blank slns did not show any distinct peak at that temperature . a distinct dcx endothermic melting peak was also absent in the dcx - slns ( figure 4a ) . on the other hand , the presence of the endothermic melting peak of trimyristin at approximately 5558 c confirmed the solid state of the lipid within the slns ( figure 4a ) . finally , xrd showed that a characteristic dcx peak was present in the physical mixture , but absent from the dcx - sln composition ( figure 4b ) . dsc thermograms ( a ) and x - ray diffractograms ( b ) of dcx - slns , dcx alone , trimyristin ( tm ) alone , blank slns , or the physical mixture of blank slns and dcx . mtt assay revealed that both dcx - slns and dcx in t80/e inhibited the proliferation of tumor cells , including murine mammary gland cancer cells ( m - wnt ) , murine lung cancer cells ( tc-1 ) , and human breast adenocarcinoma cells ( mda - mb-231 ) . however , the ic50 values of the dcx - slns were significantly lower than that of the dcx in t80/e in each of the three cell lines ( figure 5a ) . at the highest equivalent dcx concentrations tested ( i.e. , 0.01 , 1 , and 0.05 m in m - wnt , tc-1 , and mda - mb-231 , respectively ) , both the blank slns and the t80/e vehicle control did not show any significant toxicity in all three cell lines ( data not shown ) . ( a ) the ic50 values of dcx - slns and dcx in t80/e in mda - mb-231 , m - wnt , and tc-1 cells . cells were incubated with the dcx formulations for 72 h. ( b ) caspase 3 activity ( in nmol amc / min / ml ) in tc-1 cells following 72 h of incubation with dcx - slns or dcx in t80/e ( dcx , 0.01 m ) . caspase 3 activity was also measured in tc-1 cells treated with dcx - slns , dcx in t80/e , blank slns , and t80/e at a dcx concentration of 0.01 m . caspase 3 activity in cells treated with the dcx - slns was significantly higher than in cells treated with dcx in t80/e ( p < 0.005 , figure 5b ) . the total protein contents in the cell lysates in all four groups were not significantly different ( data not shown ) . the antitumor activity of dcx - slns was evaluated in tc-1 murine lung cancer model pre - established in c57bl/6 mice . as shown in figure 6a , both dcx - slns and dcx in t80/e significantly inhibited the growth of the tc-1 tumors in mice . however , the dcx - slns were significantly more effective than the dcx in t80/e formulation , starting on day 15 ( figure 6a ) . the average body weights of mice that were injected with blank slns or 5% mannitol ( as a vehicle control ) increased slightly ( 10% ) during the 21 days after tumor cell implantation , while the average body weight of mice that were treated with dcx - slns or dcx in t80/e did not show any significant change ( figure 6b ) . finally , the average weight of tumors in mice that were treated with the dcx - slns was also significantly lower than that in other groups at the end of the study ( figure 6c ) . anti - cd31 staining ( i.e. , angiogenesis marker ) showed that the extent of cd31 staining tended to be lower in tumors in mice that were treated with the dcx - slns , as compared to in other groups ( figure 6d ) . ( a ) the growth curves of tc-1 tumors in c57bl/6 mice ( p < 0.05 , dcx - slns or dcx in t80/e vs 5% mannitol , p < 0.05 , dcx - slns vs dcx in t80/e ) . ( b ) ( d ) representative images of tumor tissues after anti - cd31 staining ( bar = 100 m ) . mice were iv injected with dcx - slns or dcx in t80/e at a dcx dose of 15 mg / kg via the tail vein on days 6 , 9 , and 12 after tumor implantation . figure 7 shows the concentration of dcx in tumors and other organs in tc-1 tumor - bearing mice 2 and 12 h after the mice were injected intravenously with either dcx - slns ( prepared with trimyristin ) or dcx in t80/ethanol . the concentration of dcx in tumors in mice that were injected with dcx - slns was about 50% higher than in mice that were injected with the dcx in t80/ethanol formulation , 12 h after the injection ( p < 0.05 ) ( figure 7a ) . however , the concentration of dcx in the liver , spleen , kidneys , heart , and lungs of mice that were injected with the dcx - slns were lower than in mice that were injected with the dcx in t80/e formulation ( figures 7b f ) . finally , 2 h after intravenous injection , the concentration of dcx in the plasma in mice that were injected with the dcx - slns was about 5-times higher than in mice that were injected with the dcx in t80/e formulation ( figure 7 g ) . the concentrations of dcx in tumor ( a ) , liver ( b ) , lungs ( c ) , heart ( d ) , kidneys ( e ) , spleen ( f ) , and plasma ( g ) of tc-1 tumor - bearing c57bl/6 mice 2 or 12 h after iv injection of either dcx - slns or dcx in t80/e . since the discovery of taxanes in the mid-1980s , the fervent search for more efficacious and less toxic taxane formulations has led to the fda approval of three market products , namely , taxol ( bristol - myers squibb , princeton , nj , usa ) , taxotere ( sanofi - aventis u.s . llc , bridgewater , nj , usa ) , and abraxane ( celgene corporation , summit , nj , usa ) . in addition , a polymeric pegylated micelle formulation of paclitaxel ( genexol - pm ) has been marketed in south korea since 2007 , and others ( e.g. , bind-014 ) are in the pipeline . taxotere used to be the only fda - approved dcx formulation on the u.s . include formulations launched by hospira , sagent , and accord , which are all dcx in tween 80/ethanol solutions . docefrez is a lyophilized dcx powder to be reconstituted with 3.54% ethanol in tween 80 before injection . dcx has low water solubility ( 5 g / ml ) , and tween 80 and ethanol are used in the current dcx formulations to solubilize dcx . the main aim in the present study was to rationally design a tween 80-free formulation of dcx that also improves the antitumor activity of dcx . , where the authors reported that dcx solubility in low melting point triglycerides , such as tributyrin , tricaproin , and tricaprylin , is 10,00020,000 times more than in water , we postulated that triglyceride - based sln formulations may exhibit attractive drug four different medium- and long - chain saturated triglycerides that are solid at body temperature were employed in this study ; namely , trilaurin ( mp 46 c ) , trimyristin ( mp 57 c ) , tripalmitin , ( mp 66 c ) , and tristearin ( mp 69 c ) with fatty acid chain lengths of 12 ( c12:0 ) , 14 ( c14:0 ) , 16 ( c16:0 ) , and 18 ( c18:0 ) carbon atoms , respectively . it was reported that triglycerides with melting points lower than room temperature form nanodroplets that are prone to coalescence during preparation or storage . the use of high melting point triglycerides may significantly decrease the mobility of the drug molecules within the lipid core and thus reduce immature drug leakage . therefore , tricaprylin ( c8:0 , mp 9 c ) , tricaprin ( c10:0 , mp 31 c ) , and trolein ( c18:1 , mp 5 c ) were excluded . the trimyristin - based sln formulation was chosen from the four tested formulations because the rate at which the dcx was released from them was the slowest ( figure 1 ) , and the slns were also more stable ( figure 2 ) . in a mouse model with pre - established tc-1 mouse tumors , the dcx - slns were significantly more effective than dcx in t80/e in inhibiting the tumor growth ( figure 6 ) , likely because the dcx - slns significantly increased the accumulation of dcx in tumor tissues ( figure 7a ) . circulating nanocarriers usually take advantage of the leaky vasculature and/or poor lymphatic drainage in tumor tissues to accumulate in tumors . since dcx accumulation in tumor was only significantly higher at 12 h , but not at 2 h , following the administration of slns , as compared to the dcx in t80/e , it is likely that the enhanced retention of the dcx - slns in tumors due to poor lymphatic drainage was responsible for the improved accumulation . small molecules may also exhibit enhanced permeation to tumors as well due to the leaky neovasculature in the tumor tissues , however , they can easily diffuse out of tumors as well , while macromolecules and nanocarriers are entrapped and consequently accumulate . dcx not only induces apoptosis due to microtubule assembly stabilization , but also is antiangiogenic . inhibition of the formation of new blood vessels that provide rapidly growing tumors with increasing nutritional demand is expected to stunt the tumor growth . furthermore , the hastily formed tumor - associated vasculature is characterized with various fenestration and imperfections , and almost lacking any intact lymphatic drainage . nanoparticles are known to take advantage of this leaky tumor vasculature to extravasate into the tumor microenvironment , where they can accumulate . the most widely used lipid excipients are soy lecithin , glycerides , or a mixture of the two . formulated dcx into a nanostructured lipid carrier based on soy lecithin , glyceryl monostearate , and fatty acids , but the dcx in nanostructured lipid carrier was only slightly more effective than a dcx in a t80/e formulation ( duopafei ) against b16 tumors in mice . xu et al . designed a trimyristin - based sln formulation for dcx to treat hepatocellular carcinoma instead , their surface was galactosylated to target asialoglycoprotein receptors overexpressed on the surface of hepatoma cell lines . we previously reported the formulation of dcx - loaded lecithin - based pegylated slns . the resultant slns showed an improved in vitro cytotoxic activity , in addition to improved tumor accumulation in a mouse model . however , the capacity of this formulation in incorporating dcx was limited , which may be attributed to the limited affinity between dcx and the excipients . in the present study , in order to rationally select the most suitable excipient , four dcx - sln formulations were prepared using four different high melting point triglycerides . as discussed previously , a successful formulation for taxane is the one that exhibits ( a ) long plasma circulation time , ( b ) long drug retention within the delivery carrier , which requires high drug excipient affinity and slow release , ( c ) high tumor accumulation , and ( d ) favorable biodistribution profile , as less drug goes to healthy tissues . drug release behavior from the dcx - slns and short - term stability were the criteria for triglyceride selection in this study . in this regard , the absence of a burst release of dcx from the dcx - slns in the first 2 h and a subsequent slower release rate predict a limited drug leakage from the nanoparticles in the blood circulation before reaching tumors . the release of dcx from the tristearin - based and trilaurin - based dcx - slns was relatively faster ( figure 1 ) , and the tripalmitin- and trilaurin - based dcx - slns showed instability , as the zeta potential changed significantly following a short - term storage ( figure 2 ) . change in zeta potential has been used as an indicator of nanoparticle instability . several reports indicated that trilaurin does not exist in the solid state within the slns , but rather as a supercooled - liquid state that resembles o / w emulsions , even at 4 c , which was not the case with triglycerides having higher melting points . the relatively faster release of dcx from the trilaurin - based slns may also arise as a result of the same phenomenon . phenomenon was not reported , therefore , the reasons to which this sign of instability may be attributed need to be investigated . on the other hand , the absence of a burst release of dcx from the dcx - slns prepared with trimyristin may infer a strong dcx trimyristin interaction . it was reported that the solubility of dcx in tributyrin ( 4 c chain ) is about 108 mg / ml , and it gets lower with higher chain length , reaching about 56 mg / ml with tricaprylin ( 8 c chain ) . based on this , and since dsc and xrd data ( figure 4 ) implied that there is a strong interaction between dcx and the excipients , it is speculated that dcx exists in the slns in either a noncrystalline state or a dissolved state , or both , within the lipid matrix . existence of dcx in the amorphous state within lipid - based matrices was also reported previously . the disappearance of the characteristic dcx - related peaks in dsc and xrd was previously shown to be related to the loss of dcx crystallinity . in fact , we also found that the characteristic melting peak of dcx at 167 c completely disappeared upon analyzing the thermal behavior of dcx trimyristin mixtures at dcx to trimyristin ratios of 1:5 , 1:2 , and 1:1 using dsc ( data not shown ) , suggesting the prevalence of a strong interaction between the dcx and trimyristin . the relatively slower release of dcx from trimyristin - based dcx - slns may also be attributed to the strong dcx long - chain triglycerides in soybean oil and egg yolk phospholipids are commonly used in intravenous fat emulsions as components of parenteral nutrition for patients who are not able to receive nutrition via oral diets ( e.g. , intralipid , b braun medical inc . , bethlehem , pa ) . triglycerides are metabolized in the blood by lipases into corresponding fatty acids , which are cleared from the blood within about 30 min . phosphatidylcholine and pegylated phosphoethanolamine are used in products that are approved for intravenous infusion in humans ( e.g. , doxil ) . therefore , we expect that our new dcx - sln formulations will likely have a favorable safety profile . in fact , the body weights of the tumor - bearing mice that were treated with the dcx - slns did not significantly change by the end of the efficacy study ( figure 6b ) . in addition , the concentrations of dcx in vital organs such as liver , spleen , kidneys , lungs , and heart of mice that were injected with the dcx - slns were significantly lower than in mice that were injected with the dcx in t80/e formulation ( figure 7 ) , indicating that our dcx - slns may be less damaging to those vital organs than taxotere . in the present study , by taking advantage of the high solubility of dcx in triglycerides , we successfully prepared several dcx - incorporated slns using various medium- and long - chain triglycerides . the dcx - sln composition prepared with trimyristin was selected for further evaluation because the resultant dcx - slns were stable in a short - term stability study , and the rate at which the dcx was released from them was the slowest . the dcx - slns showed a stronger antitumor activity than dcx solubilized in a tween 80/ethanol mixture in cell culture and , more importantly , in a mouse model with pre - established tumors , likely because the dcx - slns significantly increased the accumulation of the dcx in tumor tissues . the decreased accumulation of dcx in vital organs after iv injection of dcx - slns , relative to after injection of dcx solubilized in a tween 80/ethanol mixture , suggests that the dcx - slns may have a favorable safety profile .

synovial sarcoma is a malignant mesenchymal neoplasm which commonly occurs in the extremities of adults , in close association with joint capsules , tendon sheaths , bursae and fascial structures . only a few cases of synovial sarcoma occurring in the abdominal wall have been reported . a case of a primary synovial sarcoma arising from the anterior abdominal wall fascial aponeurosis is presented . docetaxel ( dcx ) is a second generation taxane . it is approved by the u.s . food and drug administration for the treatment of various types of cancer , including breast , non - small cell lung , and head and neck cancers . however , side effects , including those related to tween 80 , an excipient in current dcx formulations , can be severe . in the present study , we developed a novel solid lipid nanoparticle ( sln ) composition of dcx . trimyristin was selected from a list of high melting point triglycerides as the core lipid component of the slns , based on the rate at which the dcx was released from the slns and the stability of the slns . the trimyristin - based , pegylated dcx - incorporated slns ( dcx - slns ) showed significantly higher cytotoxicity against various human and murine cancer cells in culture , as compared to dcx solubilized in a tween 80/ethanol solution . moreover , in a mouse model with pre - established tumors , the new dcx - slns were significantly more effective than dcx solubilized in a tween 80/ethanol solution in inhibiting tumor growth without toxicity , likely because the dcx - slns increased the concentration of dcx in tumor tissues , but decreased the levels of dcx in major organs such as liver , spleen , heart , lung , and kidney . dcx - incorporated slns prepared with one or more high - melting point triglycerides may represent an improved dcx formulation .

the prevalence of diabetes mellitus ( dm ) for all age groups worldwide was estimated to be 2.8% in year 2000 and it is quite clear that male patients with dm are 3 times more likely to develop erectile dysfunction ( ed ) than those who do not have dm . in streptozotocin ( stz)-induced diabetic rats , thus , the erectile process has been shown to be strongly androgen - sensitive , at least in the rat model . testosterone substitution can reverse hypogonadism and diabetes - induced penile hyporeactivity to cavernous nerve stimulation in vivo in rats , along with increased neuronal nitric oxide synthase ( nnos ) expression . insulin - like growth factor binding protein-3 ( igfbp-3 ) is a member of the growth factor family and it is generally believed that the increased expression of igfbp-3 is associated with ed in diabetic rats , while igfbp expression is suppressed by androgens . it is well known that short hairpin rna ( shrna ) is emerging as a technology to silence gene expression by inhibiting mrna translation and/or inducing its degradation , including in the penis . in the current study , we investigated whether short hairpin ribonucleic acid constructs targeting igfbp-3 ( igfbp-3 shrna ) can rehabilitate decreased testosterone concentrations in stz - induced diabetic rats . to design igfbp-3 shrna construct , the rat igfbp-3 gene sequence ( gi : m31837 ) was analyzed for a potential sirna target using the web - based sirna target finder and design tool provided on the ambion website ( ambion , inc . double - stranded sirnas ( nucleotide position : 611 ) were transcribed in vitro using the silencer sirna construction kit ( ambion ) following the manufacturer s instructions . the inhibitory sirna ( 5-gcgctacaaagttgactatga-3 , nucleotide position 611 ) was then cloned into the pgpu6/gfp / neo plasmid vector ( 2 version , ambion ) as a short hairpin dna sequence ( 5 sense strand : 5-caccgcgctacaaagttgactatgatt caagagatcatagtcaactttgtagcgcttttttg-3 ) according to the manufacturer s instructions . the pgpu6/gfp / neo- igfbp-3 shrna plasmid was purified using the endo - free maxi kit ( qiagen , valencia , ca ) . plasmids were quantitated by spectrophotometry and prepared in 0.9% saline solution at a concentration of 1.0 g/l . twenty - seven adult spf male wistar rats ( age 3 months , weight 310330 g , certificated no . scxk[e]2008 - 0005 ) were obtained from hubei laboratory animal of research center ( wuhan , china ) . the wuhan university animal care and injections of citrate buffer ( 100 mmol / l citric acid and 200 mmol / l disodium phosphate , ph 7.0 ) . rats were considered diabetic if blood glucose levels were greater than 200 mg / dl ( table 1 ) . animals received 60 l citrate buffer ( group 1 and 2 ) and 60 ul lipofectamine - plasmid complex ( 10 g / kg ) ( group 3 ) into the corpus cavernosum 12 weeks after stz induction . a constriction band was applied at the base of the penis , and the needle was left in place for 5 min to allow the medication to diffuse throughout the cavernous space . at 12 weeks after igfbp-3 shrna administration , erectile function was evaluated by measuring intracavernous pressure ( icp ) following electrostimulation of the cavernous nerves , as previously described . the ratio of maximal icp - to - mean arterial pressure ( map ) ( icp / map ) and total icp ( area under curve ) were recorded for each rat . the penile shaft was collected for further analyses and whole blood samples from both systemic and penile shaft were also collected with serum obtained after clotting for 30 min at room temperature following centrifugation . all the serum samples were immediately snap - frozen in liquid nitrogen after collection and stored at 80c for further analyses . total rna was extracted from rat penile samples using trizol reagent ( invitrogen , merelbeke , belgium ) according to the manufacturer s protocol and re - suspended in rnase - free water . total rna was stored at 80c until analysis of igfbp-3 mrna levels by myiq single - color real - time pcr detection system ( bio - rad laboratories , hercules , ca ) with the following primers : igfbp-3 , forward 5-agccgtctcctggaaacacc-3 and reverse 5-cccgctttctgcctttgg-3 ; quantitative mrna measurements were performed in triplicate and normalized to an internal control of gapdh . after the pcr program , data were analyzed with abi 7300 sds software ( applied biosystems , foster city , ca ) . the rat penile samples were lysed in ripa buffer ( 1tbs , 1% nonidet p-40 , 0.5% sodium deoxycholate , 0.1% sds , 0.004% sodium azide , 10 l / ml pmsf , 10 l / ml protease inhibitor cocktail , 10 l / ml sodium orthovanadate ) . protein concentration was measured using the coomassie plus protein assay reagent ( pierce biotechnology , rockford , il ) . equal quantities ( 30 g ) of lysates were separated on 10% sodium dodecyl sulfate gradient polyacrylamide gels and electro - blotted onto pvdf membranes ( bio - rad , hercules , ca ) . the membranes were blocked and then incubated with rabbit anti - igfbp-3 antibody ( sc-9028 ) at 1:1000 dilutions ( santa cruz biotechnology , santa cruz , ca ) . chemiluminescence was detected using ecl western blotting detection reagents ( amersham , buckinghamshire , uk ) . the expression of igfbp-3 protein was visualized by densitometry using the mivnt image analysis system ( shanghai institute of optical instruments , shanghai , china ) . the measurement of serum testosterone concentration in penile tissue was performed using an enzyme - linked immunosorbent assay ( elisa ) testosterone detection kit ( r&d systems , minneapolis , mn ) . similarly the measurement of cgmp concentration in penile tissue was performed using an enzyme - linked immunosorbent assay ( elisa ) cgmp detection kit ( r&d systems , minneapolis , mn ) . differences among normal control group , diabetic control group , and igfbp-3 shrna treatment group were considered significant at the level of p<0.05 using the 2-tailed unpaired t test . to design igfbp-3 shrna construct , the rat igfbp-3 gene sequence ( gi : m31837 ) was analyzed for a potential sirna target using the web - based sirna target finder and design tool provided on the ambion website ( ambion , inc . double - stranded sirnas ( nucleotide position : 611 ) were transcribed in vitro using the silencer sirna construction kit ( ambion ) following the manufacturer s instructions . the inhibitory sirna ( 5-gcgctacaaagttgactatga-3 , nucleotide position 611 ) was then cloned into the pgpu6/gfp / neo plasmid vector ( 2 version , ambion ) as a short hairpin dna sequence ( 5 sense strand : 5-caccgcgctacaaagttgactatgatt caagagatcatagtcaactttgtagcgcttttttg-3 ) according to the manufacturer s instructions . the pgpu6/gfp / neo- igfbp-3 shrna plasmid was purified using the endo - free maxi kit ( qiagen , valencia , ca ) . plasmids were quantitated by spectrophotometry and prepared in 0.9% saline solution at a concentration of 1.0 g/l . twenty - seven adult spf male wistar rats ( age 3 months , weight 310330 g , certificated no . scxk[e]2008 - 0005 ) were obtained from hubei laboratory animal of research center ( wuhan , china ) . the wuhan university animal care and injections of citrate buffer ( 100 mmol / l citric acid and 200 mmol / l disodium phosphate , ph 7.0 ) . rats were considered diabetic if blood glucose levels were greater than 200 mg / dl ( table 1 ) . animals received 60 l citrate buffer ( group 1 and 2 ) and 60 ul lipofectamine - plasmid complex ( 10 g / kg ) ( group 3 ) into the corpus cavernosum 12 weeks after stz induction . half the dose was administered in each crus . during intracavernosal injection , a constriction band was applied at the base of the penis , and the needle was left in place for 5 min to allow the medication to diffuse throughout the cavernous space . at 12 weeks after igfbp-3 shrna administration , erectile function was evaluated by measuring intracavernous pressure ( icp ) following electrostimulation of the cavernous nerves , as previously described . the ratio of maximal icp - to - mean arterial pressure ( map ) ( icp / map ) and total icp ( area under curve ) were recorded for each rat . the penile shaft was collected for further analyses and whole blood samples from both systemic and penile shaft were also collected with serum obtained after clotting for 30 min at room temperature following centrifugation . all the serum samples were immediately snap - frozen in liquid nitrogen after collection and stored at 80c for further analyses . total rna was extracted from rat penile samples using trizol reagent ( invitrogen , merelbeke , belgium ) according to the manufacturer s protocol and re - suspended in rnase - free water . total rna was stored at 80c until analysis of igfbp-3 mrna levels by myiq single - color real - time pcr detection system ( bio - rad laboratories , hercules , ca ) with the following primers : igfbp-3 , forward 5-agccgtctcctggaaacacc-3 and reverse 5-cccgctttctgcctttgg-3 ; quantitative mrna measurements were performed in triplicate and normalized to an internal control of gapdh . after the pcr program , data were analyzed with abi 7300 sds software ( applied biosystems , foster city , ca ) . the rat penile samples were lysed in ripa buffer ( 1tbs , 1% nonidet p-40 , 0.5% sodium deoxycholate , 0.1% sds , 0.004% sodium azide , 10 l / ml pmsf , 10 l / ml protease inhibitor cocktail , 10 l / ml sodium orthovanadate ) . protein concentration was measured using the coomassie plus protein assay reagent ( pierce biotechnology , rockford , il ) . equal quantities ( 30 g ) of lysates were separated on 10% sodium dodecyl sulfate gradient polyacrylamide gels and electro - blotted onto pvdf membranes ( bio - rad , hercules , ca ) . the membranes were blocked and then incubated with rabbit anti - igfbp-3 antibody ( sc-9028 ) at 1:1000 dilutions ( santa cruz biotechnology , santa cruz , ca ) . chemiluminescence was detected using ecl western blotting detection reagents ( amersham , buckinghamshire , uk ) . the expression of igfbp-3 protein was visualized by densitometry using the mivnt image analysis system ( shanghai institute of optical instruments , shanghai , china ) . the measurement of serum testosterone concentration in penile tissue was performed using an enzyme - linked immunosorbent assay ( elisa ) testosterone detection kit ( r&d systems , minneapolis , mn ) . similarly the measurement of cgmp concentration in penile tissue was performed using an enzyme - linked immunosorbent assay ( elisa ) cgmp detection kit ( r&d systems , minneapolis , mn ) . differences among normal control group , diabetic control group , and igfbp-3 shrna treatment group were considered significant at the level of p<0.05 using the 2-tailed unpaired t test . we determined the ratio of icp / map and the total icp ( area under curve ) during electrostimulation of the cavernous nerves at 12 weeks after igfbp-3 shrna administration . the representative icp tracing in response to electrostimulation of the cavernous nerves is shown in figure 1(a c ) . electrostimulation elicited significantly increased icp / map ratio ( figure 1d ) and total icp ( figure 1e ) in the igfbp-3 shrna treatment group compared to those in the diabetic control group ( p<0.01 , respectively ) . real - time qpcr analysis using gapdh as a housekeeping gene showed that cavernous igfbp-3 mrna level in the igfbp-3 shrna treatment group was significantly lower than in the diabetic control group ( p<0.01 , figure 2 ) . accordingly , western blot analysis showed that cavernous igfbp-3 protein level in the igfbp-3 shrna treatment group was significantly lower than in the diabetic control group ( p<0.01 , figure 3 , lower ) . these results are consistent with the results of real - time qpcr analysis and prove that igfbp-3 shrna inhibits igfbp-3 expression in rat cavernous tissue . both serum testosterone and cavernous cgmp concentrations were measured by elisa and we found that serum testosterone and cavernous cgmp concentrations were both significantly increased in the igfbp-3 shrna treatment group compared to those in the diabetic control group at 12 weeks after igfbp-3 shrna administration ( p<0.01 , figure 4 ) . we determined the ratio of icp / map and the total icp ( area under curve ) during electrostimulation of the cavernous nerves at 12 weeks after igfbp-3 shrna administration . the representative icp tracing in response to electrostimulation of the cavernous nerves is shown in figure 1(a c ) . electrostimulation elicited significantly increased icp / map ratio ( figure 1d ) and total icp ( figure 1e ) in the igfbp-3 shrna treatment group compared to those in the diabetic control group ( p<0.01 , respectively ) . real - time qpcr analysis using gapdh as a housekeeping gene showed that cavernous igfbp-3 mrna level in the igfbp-3 shrna treatment group was significantly lower than in the diabetic control group ( p<0.01 , figure 2 ) . accordingly , western blot analysis showed that cavernous igfbp-3 protein level in the igfbp-3 shrna treatment group was significantly lower than in the diabetic control group ( p<0.01 , figure 3 , lower ) . these results are consistent with the results of real - time qpcr analysis and prove that igfbp-3 shrna inhibits igfbp-3 expression in rat cavernous tissue . both serum testosterone and cavernous cgmp concentrations were measured by elisa and we found that serum testosterone and cavernous cgmp concentrations were both significantly increased in the igfbp-3 shrna treatment group compared to those in the diabetic control group at 12 weeks after igfbp-3 shrna administration ( p<0.01 , figure 4 ) . in the present study , we found that concentration of serum testosterone was significantly higher in the igfbp-3 shrna treatment group compared to that in the diabetic control group ( p<0.01 ) at 12 weeks after igfbp-3 shrna administration . these results suggest that igfbp-3 shrna may rehabilitate erectile function via increases of concentrations of serum testosterone and cavernous cgmp in stz - induced diabetic rats . the possibility that hypogonadism might contribute to diabetes mellitus ( dm)-related ed has been reconsidered recently , because of a higher prevalence of low testosterone in diabetic than in nondiabetic individuals [ 1012 ] , and even in animal models of chemical diabetes , overt hypogonadism is often reported . essentially , androgens play a dual role in the erectile process by controlling both pro - erectile and anti - erectile signaling pathways . on the one hand , androgens exert a positive role in the maintenance of the general architecture of the penile contractile structure . castration induces cavernous smooth muscle atrophy and contributes to structural alteration of erectile tissue , with a relative increase in connective tissue content [ 1618 ] . furthermore , castration reduced nitric oxide ( no ) and its generating enzyme no synthase ( nos ) , as well as nos - containing nerve fibers . thus , in castrated rats , erectile response is markedly impaired , but testosterone ( t ) reverses this deficiency . a recent study reported that testosterone may prevent impaired erectile function by maintaining at the control level the expression of the key enzymes involved in the erectile process , such as nnos . on the other hand , it has been recently demonstrated that androgens up - regulate the major mechanism responsible for cgmp degradation , phosphodiesterase type 5 ( pde5 ) , and pde5 regulates the amount of cgmp available for cavernosal smooth muscle relaxation and penile erection . a clinical report demonstrated a null effect of hypogonadism on penile erection , probably because androgen reduction produces both decreased cgmp formation ( effect on no ) and degradation ( effect on pde5 ) . the major subcellular mechanism by which erection occurs involves no - induced activation of soluble guanylyl cyclase ( sgc ) and increased cgmp concentration . a significantly decreased level of cgmp in diabetes rats was associated with ed in our previous study . here , we found that serum testosterone and cavernous cgmp concentrations were both significantly increased in the igfbp-3 shrna treatment group compared to those in the diabetic control group at 12 weeks after igfbp-3 shrna administration these results suggest that igfbp-3 shrna may rehabilitate erectile function via increased concentration of serum testosterone and improvement of no - cgmp signaling activities in stz - induced diabetic rats . igfbp-3 , conventionally thought to inhibit growth through ligand sequestration , might also be antiproliferative and proapoptotic through actions independent of the insulin - like growth factor-1 ( igf-1 ) receptor . a previous study has shown that igfbp-3 is significantly correlated with erectile function in the diabetic rat model . the increased expression of igfbp-3 might lead to depletion of the smooth muscle density and elicit ed in patients with diabetes mellitus ( dm ) . since igfbp expression is suppressed by androgens , an increase of concentration of serum testosterone might decrease the expression of igfbp-3 ( the most abundant igfbp ) . thus , hyperglycemia in combination with increased igfbp-3 expression may have a role in the development of ed . the current study indicated that igfbp-3 shrna may rehabilitate erectile function via increased concentration of serum testosterone and improvement of no - cgmp signaling activities in stz - induced diabetic rats . further investigation of the mechanisms by which increased concentration of serum testosterone leads to activation of no - cgmp signaling will enrich our understanding of the pathogenesis of ed of stz - induced diabetic rats . the plant antiaris toxicaria ( family moraceae ) is an indigenous plant common in ghanaian forests . foto or kyenkyen in akan and the bark cloth tree in english . despite considerable advancements in the treatment of neurological disorders currently available antiepileptic drugs ( aeds ) have debilitating adverse effects on cognition and behaviour . these adverse effects are commonly and consistently observed with barbiturates , benzodiazepines , and topiramate [ 4 , 5 ] . these problems are known to prevail more in developing countries due to lack of facilities for proper diagnosis and treatment along with monitoring of aed serum levels [ 6 , 7 ] . natural products and plants already used in traditional medicine can be a good place to start in the search for safer and more effective options . numerous plants used for the treatment of epilepsy traditionally have been shown to be potent in models of epilepsy and several such plants remain to be scientifically validated . leonotis leonurus , delphinium denudatum , mimosa pudica , and synedrella nodiflora are but a few examples [ 810 ] . preliminary screening of the aqueous extract of antiaris toxicaria revealed significant anticonvulsant effect in pentylenetetrazole - induced seizure test . hence , this study seeks to further explore the extract 's potential as an anticonvulsant . stem bark of antiaris toxicaria was harvested from the knust campus , kumasi , and authenticated at the pharmacognosy department of the faculty of pharmacy , knust , kumasi , ghana . a voucher specimen ( knust / hm1/011/s007 ) dried stem bark was milled into powder using a commercial grinder . the coarse powder ( 431 g ) was extracted by cold maceration with distilled water as solvent at room temperature for five days . filtrate was oven - dried to obtain a yield of 23.40% w / w of antiaris toxicaria aqueous extract ( aae ) . male icr mice and sprague - dawley rats weighing between 20 and 25 g and 120 and 145 g , respectively , were obtained from noguchi memorial institute for medical research , accra , ghana , and kept in the departmental animal house . animals were maintained under laboratory conditions of temperature , humidity , and light , housed in stainless steel cages ( 34 47 18 cm ) , and allowed access to water and food ad libitum . all animals were handled in accordance to the guide for the care and use of laboratory animals and experiments were approved by the faculty ethics committee . diazepam ( dzp ) , pentylenetetrazole ( ptz ) , picrotoxin ( pct ) , 4-aminopyridine ( 4-ap ) , and strychnine ( str ) were purchased from sigma - aldrich inc . , st . flumazenil was obtained from app pharmaceuticals , llc , schaumburg , il , usa . the method as described by vellucci and webster , 1984 , and moezi et al . , 2011 , the plant extract was administered at doses of 200 , 400 , and 800 mg kg body weight orally . intraperitoneal ( i.p ) injection of diazepam ( 0.1 , 0.3 , and 1 mg kg ) was used as reference anticonvulsant drug . animals were pretreated with the plant extract thirty minutes and diazepam fifteen minutes before administration of pentylenetetrazole ( ptz ) 85 mg kg subcutaneously . control animals were pretreated with distilled water ( 10 ml kg , p.o . ) . the onset of , total duration as well as frequency of clonic seizures were measured within a thirty minute period . animals received aae at doses of 200 , 400 , and 800 mg kg ( p.o . ) body weight . picrotoxin ( pct ) was injected intraperitoneally at a dose of 3 mg kg fifteen minutes after pretreatment with diazepam and thirty minutes after aae . diazepam ( 0.1 , 0.3 , and 1 mg kg ) served as positive control . control animals received distilled water ( 10 ml kg , p.o . ) . anticonvulsant activity was scored similarly to that stated in the ptz test [ 13 , 15 ] . tonic convulsions of hind limb extremities of mice were induced using electrical current ( 50 ma , 60 hz , and 0.2 seconds ) via ear clip electrodes . control group animals received distilled water orally ( 10 ml kg , p.o . ) . carbamazepine at doses of 3 , 10 , and 30 mg kg orally served as reference anticonvulsant . aae was tested at doses of 200 , 400 , and 800 mg kg orally . aae was administered orally at doses of 200 , 400 , and 800 mg kg body weight . strychnine was injected subcutaneously at a dose of 2 mg kg thirty minutes after aae administration . standard anticonvulsant employed was diazepam ( 0.1 , 0.3 , and 1 mg kg , i.p ) . the onset of , decrease in the total duration plus frequency of tonic convulsions were taken as indication of anticonvulsant activity . animals received aae orally at doses of 200 , 400 , and 800 mg kg body weight . 4-aminopyridine was dissolved in normal saline and injected subcutaneously at a dose of 10 mg kg body weight thirty minutes after drug treatments . control animals were pretreated with normal saline ( 10 ml kg ) and sodium valproate at 100 , 200 , and 400 mg kg served as positive control . hind limb tonic extensions and decrease in the total duration of convulsions were recorded . method as described by morales - villagrn and tapia , 1996 , was used with slight modifications . in order to investigate the mechanism of action of aae as an anticonvulsant , mice were treated with flumazenil , a benzodiazepine antagonist ( 1 mg kg , i.p ) fifteen minutes before the administration of aae ( 400 mg kg , p.o . ) . thirty ( 30 ) minutes later , convulsions were induced with pentylenetetrazole at 85 mg kg , intraperitoneally . other groups of animals received aae or flumazenil or diazepam ( 0.3 mg kg , i.p ) only . animals were observed for thirty minutes after treatment via video recording for latency and duration of convulsions . analysis of variance ( anova ) followed by newman - keuls ' post hoc test was used to determine significant differences between means . two - way anova followed by bonferroni test was used in the flumazenil experiment . in the 4-aminopyridine seizure test , the kaplan - meier method was used in estimating survival relative to time and survival differences were analyzed with the log - rank test . statistical analyses were carried out with graph pad prism version 5.0 ( graphpad software , san diego , ca , usa ) and sigmaplot version 11.0 ( systat software , inc . , pentylenetetrazole ( 85 mg kg , s.c . ) produced myoclonic jerks in all mice pretreated with the distilled water . aae ( 200800 mg kg ) produced a significant ( p = 0.0021 ; f3,16 = 7.671 ; figure 1(a ) ) dose - dependent increase in time taken to the onset of clonic seizures . in the extract - treated animals , frequency of seizures was also decreased nondose - dependently , and only the 800 mg kg dose was significant ( p = 0.0511 ; f3,16 = 3.213 ; figure 1(a ) ) . aae produced significant ( p = 0.0018 ; f3,16 = 8.005 ; figure 1(c ) ) dose - dependent decrease in the total duration of convulsions in all animals pretreated with the various doses of the extract . the reference anticonvulsant diazepam ( 0.11.0 mg kg , i.p ) also profoundly delayed the onset of myoclonic jerks , decreased frequency of jerks and significantly ( p < 0.0001 ; f3,16 = 125.8 ; figures 1(b ) and 1(d ) ) antagonized ptz - induced seizures . picrotoxin ( 3 mg kg , i.p ) produced clonic seizures in all mice pretreated with the distilled water . aae ( 200800 mg kg ) produced a dose - dependent increase in latency to seizures which was significant ( p = 0.0009 ; f3,16 = 9.108 ; figure 2(a ) ) at all doses . the frequency of seizures was also significantly ( p = 0.0081 ; f3,16 = 5.591 ; figure 2(a ) ) decreased dose - dependently . aae produced significant ( p = 0.0002 ; f3,16 = 12.53 ; figure 2(c ) ) dose - dependent decrease in the total duration of seizures in all animals . the reference anticonvulsant diazepam ( 0.11.0 mg kg , i.p . ) also profoundly delayed the onset of myoclonic jerks , decreased frequency of jerks and significantly antagonised ( p < 0.0001 ; f3,16 = 17.72 ; figures 2(b ) and 2(d ) ) pct - induced seizures . the extract did not affect the latency to onset of hind limb tonic extensions and duration of convulsions significantly ( figure 3 ) . the reference anticonvulsant carbamazepine ( 3 , 10 , and 30 mg kg , p.o . ) , however , delayed the onset of hles significantly ( p = 0.0054 ; f3,16 = 6.193 ; figure 3 ) and decreased the total duration of electroshock - induced convulsions . aae did not affect the latency to onset of hind limb tonic extensions and duration of convulsions significantly ( figure 4 ) . the reference anticonvulsant diazepam ( 1 mg kg , i.p ) , on the other hand , delayed the latency to convulsions significantly ( p < 0.0001 ; f4,20 = 36.60 ; figure 4 ) . the duration of convulsions was also significantly ( p < 0.0001 ; f4,20 = 14.31 ; 4-aminopyridine ( 10 mg kg , i.p . ) produced hind limb tonic extensions in all animals . the extract produced a significant ( p < 0.0001 ; f3,28 = 22.02 ; figure 5(a ) ) increase in time taken to the onset of convulsions . sodium valproate ( 100400 mg kg ) also significantly ( p < 0.0001 ; f3,28 = 17.61 ; figure 5(b ) ) delayed the onset of convulsions . the extract significantly ( p = 0.0004 , (df = 3 ) = 18.11 ; figure 6(a ) ) improved survival of the animals after induction of convulsions . the test for trend presented significant ( p < 0.0001 , (df = 1 ) = 15.79 ) effect of the treatment groups on median survival indicating a linear trend . sodium valproate also produced similar effects on survival ( p < 0.0001 , (df = 3 ) = 21.23 ; figure 6(b ) ) and linear trends ( p < 0.0001 , (df = 1 ) = 19.17 ) . survival curves show a decrease in probability of survival with time . however , probability of survival increased with increasing dose . flumazenil significantly ( p = 0.0009 ; f1,8 = 26.09 ; figure 7(c ) ) reversed the reduction in duration of seizures produced by the extract . a significant reversal ( p = 0.0003 ; f1,8 = 37.51 ; figure 7(a ) ) was also obtained for the latency to seizures . effects of diazepam were significantly ( p < 0.0001 ; figures 7(a ) , 7(b ) , and 7(c ) ) reversed by flumazenil . the outcome of this study provides evidence that the aqueous extract of the stem bark of antiaris toxicaria possesses anticonvulsant activity . the ability of aae to delay the onset of convulsions and/or shorten the duration of convulsions was considered an indication of anticonvulsant activity . judging from the data obtained , the plant extract exhibited anticonvulsant activity in the ptz test just like diazepam which can be due to action on gaba system [ 1921 ] . it , however , has been shown to be due to inhibition and/or attenuation of gabaergic neurotransmission [ 22 , 23 ] . it is therefore likely that aae produces its anticonvulsant effect directly or indirectly by enhancing gabaergic neurotransmission in the brain . - synaptic inhibition mediated by gaba often leads to inhibition of neurotransmitter release from the excitatory arm . pct - induced seizures which are due to the decreased gabaa - receptor - mediated inhibition in turn promote the excitatory arm of the cns mainly mediated by glutamate [ 20 , 22 ] . the extract being effective in the pct - induced seizure test points to a more specific action on gaba - mediated neurotransmission . this has been further confirmed by the use of flumazenil to reverse the anticonvulsant properties of aae in the ptz - induced seizure model . flumazenil is a benzodiazepine antagonist at the gabaa receptor possessing the ability to reverse anticonvulsant effects and the accompanying alterations in extracellular glutamate concentration . the specificity of flumazenil to this receptor has been proven as several authors have demonstrated that the antagonist has no effect on diazepam 's action on voltage - dependent na channels [ 2931 ] . retigabine is currently the only approved drug for the treatment of epilepsy which functions through activation of potassium currents [ 3234 ] and has promise in the treatment of pharmacoresistant epilepsy . it is also known that 4-aminopyridine stimulates the release of neurotransmitters , including glutamate , in numerous preparations such as brain slices [ 3538 ] and the neuromuscular junction . furthermore , it has been recently found by a microdialysis procedure in vivo that glutamate is by far the amino acid predominantly released by 4-aminopyridine in the striatum . therefore , due to the well - recognized function of excitatory amino acid receptors in convulsive and excitotoxic mechanisms , it seems feasible that the excess glutamate released may be involved in the excitatory effects . this indicates that the extract may be acting , in addition to the above , by modulating the release of glutamate . from the pattern of survival curves , it can be inferred that the dose of the extract does affect survival rate offering significant protection against death from 4-aminopyridine - induced convulsions . this was evident from the log - rank analysis which indicated significant differences between survival curves for each dose level . median survival time ranged from 30 min and death may be prevented totally at the highest dose of aae . the exact mechanism by which the extract offers this protection may be linked to its ability to reduce excitotoxic effects mediated by glutamate release . this indicates that it is not effective in partial and generalized tonic seizures and is unable to prevent seizure spread . the fact that aae produced no protective effects against strychnine - induced convulsions suggests it does not interact with the glycine - mediate inhibitory pathway . the results presented here indicate that the aqueous extract of antiaris toxicaria exhibits anticonvulsant activity , possibly through gaba - mediated inhibition and inhibition of glutamate mediated excitation via activation of potassium ion channels .

backgroundthe aim of this study was to determine if shrna constructs targeting insulin - like growth factor binding protein-3 can rehabilitate decreased serum testosterone concentrations in streptozotocin - induced diabetic rats.material/methodsafter 12 weeks of intracavernous administration of igfbp-3 shrna , intracavernous pressure responses to electrical stimulation of cavernous nerves were evaluated . the expression of igfbp-3 at mrna and protein levels was detected by quantitative real - time pcr analysis and western blot , respectively . the concentrations of serum testosterone and cavernous cyclic guanosine monophosphate were detected by enzyme - linked immunosorbent assay.resultsafter 12 weeks of intracavernous administration of igfbp-3 shrna , the cavernosal pressure was significantly increased in response to the cavernous nerves stimulation compared to the diabetic control group ( p<0.01 ) . cavernous igfbp-3 expression at both mrna and protein levels was significantly inhibited . both serum testosterone and cavernous cyclic guanosine monophosphate concentrations were significantly increased in the igfbp-3 shrna treatment group compared to the diabetic control group ( p<0.01).conclusionsthese results suggest that igfbp-3 shrna may rehabilitate erectile function via increases of concentrations of serum testosterone and cavernous cyclic guanosine monophosphate in streptozotocin - induced diabetic rats . antiaris toxicaria ( moraceae ) was evaluated for anticonvulsant activity in rodents . animal models used include maximal electroshock test ( mest ) ; pentylenetetrazole - induced ( ptz ) convulsions ; picrotoxin - induced ( pct ) convulsions ; strychnine- ( str- ) and 4-aminopyridine - induced convulsions . increase in latency to seizures as well as reduction in duration and frequency of seizures indicated anticonvulsant activity . the extract was more effective in all models used except the maximal electroshock test and strychnine - induced convulsions . antiaris toxicaria aqueous extract ( 200 , 400 , and 800 mg kg1 ) significantly ( p < 0.05 0.01 ) shortened the duration of convulsions in ptz- and pct - induced seizures . delay in the onset of convulsions in the two tests was significant ( p < 0.001 ) . reduction in the frequency of seizures was also significant ( p < 0.05 0.001 ) in both tests . antiaris further delayed the onset of seizures in 4-aminopyridine model while producing 75% protection against death in mice . diazepam ( 0.1 , 0.3 , and 1 mg kg1 ) , carbamazepine ( 3 , 10 , and 30 mg kg1 ) , and sodium valproate ( 100400 mg kg1 ) were used as reference anticonvulsant drugs for various models . flumazenil blocked the effect of the extract in the ptz test significantly suggesting that antiaris toxicaria may be acting by enhancing the effects of the gabaergic system . antiaris toxicaria aqueous extract therefore possesses anticonvulsant activity .

a 15-year - old male , who was diagnosed with prs , was scheduled for repair of a pectus excavatum under general anesthesia . he had a history of tracheostomy at birth , a cleft palate operation at 1 month of age , and tracheostomy closure 1.5 years later . according to the consulting otolaryngologist , there was no pathological condition except sleep apnea . at the physical examination of his airway , the boy had the typical appearance of prs . he also had a mouth opening limitation and the maximum distance between incisors was 2.8 cm . his mallampati class was grade 3 , but he had no limited neck movement . because his parents disagreed with a conscious fiber - optic intubation for fear that their son would have a hard time during the procedure , we planned to induce anesthesia with propofol and succinylcholine and then intubate with the mcgrath mac video laryngoscope and a malleable stylet . we also prepared a laryngeal mask airway ( lma ) in case of difficult mask ventilation , according to the difficult airway guidelines . finally , if all of these methods failed , we planned to wake him because his parents wanted to cancel the surgery if the intubation failed . anesthesia was induced in the operating room with 2 mg / kg propofol and 1 mg / kg succinylcholine after preoxygenation with 100% oxygen . after full muscular relaxation , the mcgrath mac video laryngoscope was inserted into the oropharynx and was placed posterior to the epiglottis because the epiglottis obscured the glottis and provided a half view of the glottis ( cormack - lehane grade 2 ) . however , insertion of the 60 angled malleable styletted ett failed because the tube tip could be seen posterior to the arytenoids despite the external laryngeal maneuver . the tube tip reached the glottis , but there was considerable resistance advancing the ett through the vocal cords even though the stylet was withdrawn . finally , the frova intubating introducer with its tip modified as much as a 60 angle , similar to the mcgrath mac video laryngoscope , was inserted through the vocal cords ( figs . 1 and 2 ) . the frova intubating introducer was used as a railroad for the ett , and there was no difficulty advancing the ett under vision with the video laryngoscope ( fig . correct placement of the ett was further confirmed by auscultation and end - tidal co2 detection . we applied the mcgrath mac video laryngoscope and frova intubating introducer for endotracheal intubation in this case of a difficult airway and showed that the strategy was effective and successful . fiber - optic intubation is currently the gold standard for elective difficult airway management , but alternatives are described in the literature . the intubation techniques described include : airtraq optical laryngoscopy ( prodol meditec sa , vizcaya , spain ) , video laryngoscopy , non - fiber - optic intubation via lma , fiber - optic intubation via lma , and a paraglossal approach with tube advancement over a gum - elastic bougie . we chose a mcgrath mac video laryngoscope and malleable stylet because the mcgrath mac video laryngoscope provides a good view of the glottis , and a malleable stylet helps direct the ett to the glottis . as a precaution for a failed intubation , we also prepared a frova intubating introducer and fiber - optic bronchoscope for combined use , and an lma in case of a difficult mask ventilation according to the difficult airway guidelines . it is important to have backup airway management plans because it takes less than 10 min for patients with a difficult airway to become hypoxic . at our first attempt , insertion of the 60 angled malleable styletted ett failed because the tube tip could be seen posterior to the arytenoids . external laryngeal pressure and withdrawal of the blade to lessen tilting of the laryngeal axis and to reduce the introducing angle may have been helpful . however , these maneuvers did not work in our case . the tube tip reached the glottis , but there was considerable resistance advancing the ett through the vocal cords . this occurs from time to time , as the tube tip may strike the anterior tracheal wall because of the angle of the stylet . in this case , the operator withdraws the stylet approximately 4 cm , withdraws the video laryngoscope by 1 - 2 cm and rotates the ett slightly to facilitate passage into the trachea . we withdrew the stylet and rotated the ett to pass it into the trachea but failed . finally , the frova intubating introducer with its tip modified as much as a 60 angle like the mcgrath mac video laryngoscope was attempted , and it was inserted through the vocal cords with no difficulty . this was a remarkable result because the tip of the 60 angled malleable styletted ett , which had the same curvature as the frova intubating introducer , could be seen posterior to the arytenoids even though the external laryngeal maneuver was applied . unlike the macintosh laryngoscope , video laryngoscopes require a tilting force rather than a lifting force , and this force makes mouth opening too small to handle the ett . because the frova intubating introducer has a smaller outer diameter than that of an ett ( 4.6 mm for the frova intubating introducer vs. 8.9 mm for a # 6.5 ett ) , it is easy to handle in a narrow space , as shown in this case . thus , the frova intubating introducer with its tip modified as much as 60 reached the glottis , whereas the same 60 angled malleable styletted ett could not . although the frova intubating introducer was designed for use with a macintosh laryngoscope in the line of sight , and its tip should not be modified during macintosh laryngoscopy , modifying its tip and combined use with a video laryngoscope provides some advantages . first , the frova intubating introducer allows for easy handling in the narrow space under a video laryngoscope . second , it creates less trauma than a styletted ett . less resistance advancing the 60 angled frova intubating introducer through the vocal cords than that of a 90 angled malleable finally , preparing the frova intubating introducer is less time consuming than a fiber - optic bronchoscope , so it can be used for an unanticipated difficult intubation . the use of a video laryngoscope combined with a fiber - optic bronchoscope has been reported previously . fiber - optic bronchoscopy is a method of inserting an ett with a video laryngoscope . however , this technique has some disadvantages ; it needs more than two experienced anesthesiologists and one anesthesiologist should be familiar with the fiber - optic bronchoscope . in addition , the maintenance and preparation of a fiber - optic bronchoscope are time - consuming , limiting its use in the case of an unanticipated difficult intubation . use of a lma is another good option in patients with prs ; however , we thought it would be dangerous in this case because lung compliance might have decreased during repair of the pectus excavatum . we did not know that tracheal intubation with a macintosh laryngoscope would be a success or failure in this patient because we did not use a macintosh laryngoscope initially . sometimes tracheal intubation is attempted with a macintosh laryngoscope in anticipated difficult airway with little possibility of success , even though other methods for intubation , such as a video laryngoscope or fiber - optic bronchoscope , are available . the difficult airway management guidelines developed by the difficult airway society of the uk suggest that the initial tracheal intubation plan should not include more than four attempts . the fact that multiple attempts can lead to tissue trauma causes more difficult intubation in subsequent trials , makes mask ventilation difficult , and results in hypoxia . therefore , it is important to choose the first intubation plan carefully because fewer attempts lead to less airway trauma and increased patient safety . in conclusion , we propose that the combined use of a mcgrath mac video laryngoscope and a frova intubating introducer is a useful option for a difficult airway with an upper airway anomaly . rarely , severe cholecystitis may result in extravasation of bile through rupture of rokitansky - aschoff sinuses or mucosal ulceration causing severe fibrosis and formation of yellow intramural nodules and xanthogranulomatous reaction known as xanthogranulomatous cholecystitis ( xgc ) [ 1 - 4 ] . although there is a known association between gallbladder cancer and xgc , it is not clearly understood . computed tomography ( ct ) has recently become the gold standard investigation of choice in acute abdomen because it is quick , noninvasive , and provides a detailed map of the abdomen . furthermore , the introduction of multislice ct has increased the diagnostic accuracy of intra - abdominal conditions . however , ct may also detect incidental silent conditions requiring urgent attention such as abdominal aortic aneurysm ( aaa ) . we herein present a rare case of a patient who presented with signs of acute cholecystitis and gallbladder empyema . investigations showed evidence of chronic cholecystitis and an incidental large aaa . at surgery , a suspicious small nodule ( not seen on ct ) was excised . this rare coexistence of pathologies adds a further dimension to the management dilemma of the multidisciplinary team . a 79-year - old woman presented to the emergency department with a 5-week history of intermittent right upper quadrant and epigastric pain . her past medical history included hypertension , myocardial infarction 10 years previously , and bilateral knee replacement . on examination , she had tachycardia of 100 beats per minute with no fever . abdominal examination revealed a tender mass in the right upper quadrant with a pulsatile epigastric mass . a full blood count and serum biochemistry showed a hemoglobin level of 11.2 g / dl , leukocytosis of 15 10/l , c - reactive protein level of 141 mg / l , and normal renal and liver function tests . an urgent ct scan of the abdomen showed evidence of severe cholecystitis with a distended , thick - walled gallbladder , suspected gallbladder empyema , and an incidental 7-cm infrarenal aaa ( fig . 1a ) . immediate percutaneous decompression of the gallbladder empyema with a radiologically placed drain revealed 240 ml of pus . the patient 's condition improved , and she was discharged home . in view of the adjacent aaa , the decision of the multidisciplinary team was to carry out open cholecystectomy followed by staged endovascular aneurysm repair . at surgery , the gallbladder was adherent to the surrounding tissue with dense liver , duodenal , and bowel adhesions . a small nodule ( not clearly seen on ct ) was found adjacent to the distended gallbladder . xgc is a rare form of chronic cholecystitis , accounting for 0.7% to 13.2% of resected gallbladder specimens . it is thought that chronic inflammation leads to mucosal ulceration and rupture of rokitansky - aschoff sinuses with extravasation of lipids from bile , resulting in a xanthogranulomatous reaction within the gallbladder wall characterized by formation of multiple intramural yellow nodules ( fig . this inflammatory process is often extensive and may extend to adjacent organs , forming dense adhesions with a large mass of inflammatory tissue surrounding the gallbladder . the association between xgc and gallbladder cancer has been shown in the literature in small case series and some single case reports . it is estimated that xgc and gallbladder cancer coexist in up to 12% of cases . xgc is often mistaken for carcinoma of the gallbladder both macroscopically during surgery and on ct examinations ; thus , radiological differentiation from cancer can be extremely difficult in the presence of severe inflammation . furthermore , the fact that xgc can be associated with gallbladder cancer makes the differentiation even more difficult . in our patient , the clinical presentation suggested a diagnosis of acute cholecystitis and gallbladder empyema , as confirmed on ct ( fig . therefore , simple cholecystectomy was carried out as planned ; however , advanced gallbladder cancer was discovered during surgery . this nodule was less than 1 cm in size and was not shown on ct because of the extensive inflammation surrounding the gallbladder . diagnosis of xgc on ct can be difficult ; however , the presence of cholelithiasis , thickening of the gallbladder wall of > 3 mm , hypodense nodules within the gallbladder wall , and contrast enhancement of the mucosa with a distinctive presence of a hypodense band around the gallbladder are highly suggestive of xgc . kim et al . suggested that the hypodense nodules may represent abscesses of xanthogranulomas . in our patient following percutaneous drainage of the gallbladder empyema , a second ct scan showed that these hypodense areas remained unchanged , confirming that these areas corresponded to the yellow nodules seen macroscopically in the resected specimen ( fig . laparoscopic cholecystectomy is often associated with conversion to open surgery in 80% of cases . with the increased use of ct as a gold standard imaging modality in abdominal pain , more and more incidental pathologies are identified , some of which prompt urgent attention ( e.g. , aaa ) . the increase in the aging population has led to a rise in the prevalence of aaa coexisting with other nonvascular pathologies . it is estimated that 3.5% of patients have an aaa coexisting with intra - abdominal nonvascular disease . the management of concomitant pathology can present a real challenge to the multidisciplinary team , especially with large aneurysms . it is unclear whether performing simultaneous procedures or a staged approach results in a better outcome . a study by fry and fry concluded that the risk of simultaneous biliary and aortic procedures may subject the patient to a major risk of graft infection and death . they suggested that a staged operation can be performed safely if the time between the cholecystectomy and subsequent aortic reconstruction is less than 4 months . another report by thomas et al . supports the above findings . according to the above studies , the mortality rate due to performance of cholecystectomy in a single operation with aaa repair can reach to 9% . even when considering a staged procedure , the order of treatment of both pathologies poses a difficult surgical challenge .

patients with pierre robin syndrome are characterized by micrognathia , retrognathia , glossoptosis , and respiratory obstruction and are prone to have a difficult - to - intubate airway . the mcgrath mac video laryngoscope provides a better view of the glottis than a macintosh laryngoscope , but it is not easy to insert an endotracheal tube through the vocal cords because a video laryngoscope has a much greater curvature than that of a conventional direct laryngoscope and an endotracheal tube has a different curvature . the frova intubating introducer is used as a railroad for an endotracheal tube in cases of a difficult airway . we thought that a combination of these two devices would make it easy to insert an endotracheal tube through the vocal cords , as a mcgrath mac video laryngoscope provides a better glottic view and the frova intubating introducer is a useful device for placing an endotracheal tube through the glottis . we report a successful endotracheal intubation with use of the mcgrath mac video laryngoscope and frova intubating introducer in a patient with pierre robin syndrome . there have been reports of the coexistence of abdominal aortic aneurysm ( aaa ) with intra - abdominal malignancy including gastric , colonic , pancreatic , and renal . we herein report a case of a previously undiagnosed aaa and a presenting complaint consistent with acute cholecystitis . following cholecystectomy , this was noted to be a rare form of chronic cholecystitis : xanthogranulomatous cholecystitis . there is a known possible association of this uncommon condition with gallbladder cancer . the management of concomitant pathologies can present a real challenge to the multidisciplinary team , especially with large aneurysms .

neuro - cardiogenic interaction has been known for many years.1)2 ) an excessive release of catecholamines , known as a trigger of takotsubo cardiomyopathy ( tc ) , is developed when sympathetic nervous system is hyperactivated in stressful conditions including cerebral seizure . cerebral seizure has been reported as a cause of tc internationally , and cardiac complications are one of the main causes of mortality in epilepsy.3)4 ) biventricular tc is associated with more hemodynamic instability than isolated left ventricular tc . therefore , in that case , medical treatment should be more invasive and the course of hospitalization is longer.5 ) but , there has been no case report describing biventricular tc after cerebral seizure in korea . here , we report a case of a patient who presented with initial echocardiography showing biventricular apical ballooning after status epilepticus . an 83-year - old female presented to our emergency room with 2 episodes of 10 minute - duration of generalized tonic - clonic seizures at home and in the ambulance . blood pressure ( bp ) was 160/100 mm hg , pulse rate was 105/min , respiration rate was 22/min , and body temperature was 37.0. peripheral oxygen saturation was 96% with oxygen flow of 6 l / min via facial mask . she had an epilepsy 7 years prior to this attack after cerebral hemorrhage on left parietal and right temporal lobe . she was on donepezil 10 mg , methylphenidate 10 mg , choline alfoscerate 400 mg , acetyl l - carnitine 500 mg , trifrusal 300 mg because of her vascular dementia . she was independent in her activities of daily living and had never complained about chest pain or dyspnea before the admission . when she was admitted , there was another 5-minute duration of generalized tonic - clonic seizure with conjugate deviation of the eyes to the right . status epilepticus was terminated after intravenous lorazepam 4 mg injection and phenytoin 750 mg infusion . but bp was dropped from 160/100 mm hg to 70/40 mm hg and her consciousness was impaired . chest x - ray showed cardiomegaly and patchy increased opacities in right upper and lower lobes ( fig . 1 ) . arterial blood gas analysis showed a metabolic acidosis ( ph : 7.24 , pao2 : 70 mm hg , paco2 : 39 mm hg , hco3- : 16.7 mmol / l ) . a diffusion - weighted magnetic resonance brain imaging showed localized encephalomalacic lesions in left parietal lobe and right temporal pole region with peripheral old blood product deposition . division of cardiology was called for suspecting st elevation myocardial infarction ( stemi ) since an electrocardiogram ( ecg ) showed precordial v2 - 4 st segment elevation and she was found to have elevated cardiac enzymes on serial measurements ( troponin i levels 0.62 2 ) . there was no evidence of pericarditis or pheochromocytoma from her history , clinical symptoms , laboratory findings and echocardiographical features . an emergency transthoracic echocardiography ( tte ) showed an apical ballooning of the left ventricle ( lv ) with severe systolic dysfunction ( lv ejection fraction = 23% by simpsons methods ) and focal hypokinesia of right ventricular ( rv ) apex with decreased rv systolic function . fractional area change ( fac ) , [ ( rv end diastolic area - rv end systolic area ) / rv end diastolic area 100 ] was 22 % , tricuspid annular plane systolic excursion was 20 mm . despite tte finding was accordant with tc , emergency coronary angiography was done since we suspected stemi . there was no significant stenosis in left anterior descending artery , left circumflex artery and right coronary artery . however , hypokinesia of the mid to apical lv and rv from tte were discordant with coronary artery lesion . she had no further epileptic seizures during hospitalization with sodium valproate 300 mg every 12 hours and initial metabolic acidosis was resolved . after supportive care with standard heart failure therapy ( aspirin , - blocker , angiotensin converting enzyme inhibitor , 3-hydroxy-3-methylglutaryl - coenzyme a reductase inhibitor , diuretics and anti - convulsant ) , her clinical condition was getting better and moved from intensive care unit to a general ward . when patient was hospitalized for 10 days , mid to apical wall motion of lv was almost improved except for apico - anterior wall and apical rv wall motion abnormality was not observed any more ( fig . 5 ) . tc , also known as left apical ballooning syndrome or stress - induced cardiomyopathy , is named for ventricle which seems similar in appearance to a japanese octopus trap on ventriculography scan.6 ) tc is becoming well recognized as a cause of acute , reversible , and transient lv systolic dysfunction . in some cases , it would not be simple to make difference between acute coronary syndrome and tc since ecg abnormalities , elevated cardiac enzyme and symptoms mimicking stemi are common findings in tc . st - segment elevation is the most common ecg abnormality , reported in about 82% of patients , and t - wave inversion in 64%.7 ) generally in tc , there is no identifiable coronary culprit lesion explaining the wall - motion abnormality . but , as in our case , several cases of tc with coronary atherosclerotic lesion were reported.8 ) even though coronary atherosclerotic lesion was present , it was discordant with echocardiographic findings and affected regions were reversibly recovered without primary coronary intervention.9 ) in our case , there was a significant stenosis in ramus intermedius artery , however , wall motion abnormality was seen in mid to apical lv and apical rv in tte . those regional wall motion abnormalities extend beyond a single epicardial vascular distribution and discordant with coronary atherosclerotic lesion . as a result of this when seizure begins , it rises quickly within 30 min and decreases within few hours.10)11 ) a similar catecholamine release was observed in a case of tc associated with epilepsy.12 ) therefore , we can assume that seizure may trigger a condition inducing tc . in tc associated with epilepsy , complications were frequent and severe : heart failure , apical thrombus , cardiogenic shock , and left ventricular rupture were found in previous studies.13 ) although there is no hard data , it could be hypothesized that tc may be related to sudden unexpected death in epilepsy ( sudep ) . mechanisms of sudep are not clearly understood , it may include cardiac arrhythmia , myocardial ischemia , dysfunction of autonomic nervous system to the heart . cardiac abnormalities are found in up to 33% at autopsy of sudep.14 ) in our patient , apical rv hypokinesia was accompanied with lv involvement . isolated lv involvement is the most common variant , but rv involvement is becoming well recognized . it has been reported that rv involvement affects approximately 25% to 42% of patients with tc.15 ) daoko et al.5 ) said that most patients with biventricular tc are elderly women and presenting symptoms are similar to those of acute myocardial infarction . and it is known that rv involvement is associated with increasing hemodynamic instability and the risk of complications . in conclusion , tc should form a part of the differential diagnoses of cardiac event in patient with seizure . in high risk patients , such as elevated plasma troponin level and hemodynamic instability is accompanied , ecg and tte should be taken as soon as possible to detect tc and identify rv involvement . in a biventricular tc associated with epilepsy , the main causes of snhl are advanced age , the use of ototoxic medications , noise exposure , hereditary , and autoimmune diseases . it has recently been reported that various other factors such as family history , smoking , alcohol consumption , head trauma , diseases such as diabetes , high blood pressure , and some renal pathologies also lead to changes in hearing [ 25 ] . polycystic ovary syndrome ( pcos ) is a common endocrine disease affecting at least 10% of females of reproductive age . it is described by hyperandrogenism , menstrual irregularity , anovulation , infertility , and obesity . in addition , pcos is often accompanied by hyperinsulinemia , insulin resistance , dyslipidemia , and low - grade chronic inflammation that predispose to endothelial damage . also , high - frequency hearing is often affected sooner in diseases that cause endothelial damage . high - frequency ( 40008000 hz ) hearing loss was first described in pcos patients by oghan and coksuer . kucur et al . also identified eh frequency ( 800014000 ) hearing loss in pcos patients . however , these studies did not include any information about the association between hearing thresholds and the known parameters of pcos such as ir , lh / fsh ratio , hirsutism score , and free testosterone index ( fti ) . the aim of our study was to investigate the association between pcos and hearing thresholds . in addition , the relationship among insulin resistance ( ir ) , lh / fsh ratio , hirsutism score , and fti with hearing thresholds were investigated . this prospective study enrolled 80 consecutive cases that applied to the endocrinology and gynecology polyclinics of our hospital that met the inclusion criteria . forty cases ( mean age , 24.336.38 years ; range 1646 years ) that were diagnosed with pcos composed the pcos group . diagnosis of pcos was according to rotterdam eshre / asrm - sponsored pcos consensus workshop group . forty age and body mass index ( bmi ) matched female volunteers ( mean age , 26.386.75 years ; range 1642 years ) composed the control group . the study design was confirmed by the local ethics committee ( approval number : 1126.12.2012 ) and informed consent was obtained from all of the women that participated in the study . for all participants , anamnesis was obtained , and otoscopic examination and pure - tone audiometric tests were performed . the exclusion criteria were : hyperprolactinemia , congenital adrenal hyperplasia , androgen - secreting tumors , thyroid disorders , cushing syndrome , vertigo , tinnitus , middle ear diseases , major neurological and psychiatric disease , diabetes mellitus , chronic kidney failure , chronic liver failure , hypertension or hyperlipidemia and similar chronic diseases , history of previous ear surgery , family history of hearing loss , history of acoustic trauma , conductive hearing loss , subject to ototoxic substances , autoimmune diseases , cancer , exposure to radiation in the head or neck , pregnancy , smoking , alcohol use , ongoing infection or inflammation , and history of using any medication . all patients had routine audiometric tests performed by the same expert audiometrist blinded to the study . the pure - tone audiometry ( pta ) test was performed in a soundproof environment using an interacoustic clinical audiometer ( model ac40 ; denmark ) . pta - low ( 250 , 500 , 1000 , and 2000 hz ) , pta - high ( 4000 , 6000 , and 8000 hz ) and pta - extended high ( 9000 , 10000 , 11200 , 12500 , 14000 , 16000 , 18000 , and 20000 hz ) frequencies were tested . hearing threshold levels were measured in decibels ( db ) . in each case , the threshold level was calculated for both ears and at every frequency . the threshold levels at 250 hz , 500 hz , 1000 hz , and 2000 hz were identified as low - frequency audiometry , the thresholds at 4000 hz , 6000 hz , and 8000 hz as high - frequency audiometry and the thresholds at 9000 hz , 10000 hz , 11200 hz , 12500 hz , 14000 hz , 16000 hz , 18000 hz , and 20000 hz as extended - high ( eh ) frequency audiometry and the mean value was calculated . the mean hearing thresholds of the pcos group and control group at pta - low , pta - high , and pta - eh frequencies were compared . the physical examination of each of the women was performed by the same endocrinology specialist . bmi was calculated by dividing the weight by the square of height ( kg / m ) . the degree of hirsutism was determined using the ferriman - gallwey scoring ( fgs ) system . blood samples were obtained during the follicular phase of the menstrual cycle after 12 hours fasting . the fasting plasma glucose levels ( fpg ) were evaluated using roche hitachi pp modular analyzer ( roche - hitachi , tokyo , japan ) by enzymatic colorimetric assay . follicle - stimulating hormone ( fsh ) , luteinizing hormone ( lh ) , total testosterone ( tt ) , sex hormone - binding globulin ( shbg ) , and insulin measurements were done using the chemiluminescent immunoassay method with an immulite 2000 ( diagnostic products , los angeles , ca , usa ) analyzer . insulin resistance was measured using the homa - ir ( homeostasis model assessment for insulin resistance ) index . the free testosterone index ( fti ) was calculated according to the formula 100 serum testosterone ( nmol / l)/shbg ( nmol / l ) . the descriptive statistics of the variables studied were given as mean , standard deviation , and minimum and maximum values . the student t - test was used to compare data between the control group and pcos group . the statistical significance level was taken as 5% , and spss ( version 21 ) was utilized for all statistical calculations . this prospective study enrolled 80 consecutive cases that applied to the endocrinology and gynecology polyclinics of our hospital that met the inclusion criteria . forty cases ( mean age , 24.336.38 years ; range 1646 years ) that were diagnosed with pcos composed the pcos group . diagnosis of pcos was according to rotterdam eshre / asrm - sponsored pcos consensus workshop group . forty age and body mass index ( bmi ) matched female volunteers ( mean age , 26.386.75 years ; range 1642 years ) composed the control group . the study design was confirmed by the local ethics committee ( approval number : 1126.12.2012 ) and informed consent was obtained from all of the women that participated in the study . for all participants , anamnesis was obtained , and otoscopic examination and pure - tone audiometric tests were performed . the exclusion criteria were : hyperprolactinemia , congenital adrenal hyperplasia , androgen - secreting tumors , thyroid disorders , cushing syndrome , vertigo , tinnitus , middle ear diseases , major neurological and psychiatric disease , diabetes mellitus , chronic kidney failure , chronic liver failure , hypertension or hyperlipidemia and similar chronic diseases , history of previous ear surgery , family history of hearing loss , history of acoustic trauma , conductive hearing loss , subject to ototoxic substances , autoimmune diseases , cancer , exposure to radiation in the head or neck , pregnancy , smoking , alcohol use , ongoing infection or inflammation , and history of using any medication . all patients had routine audiometric tests performed by the same expert audiometrist blinded to the study . the pure - tone audiometry ( pta ) test was performed in a soundproof environment using an interacoustic clinical audiometer ( model ac40 ; denmark ) . pta - low ( 250 , 500 , 1000 , and 2000 hz ) , pta - high ( 4000 , 6000 , and 8000 hz ) and pta - extended high ( 9000 , 10000 , 11200 , 12500 , 14000 , 16000 , 18000 , and 20000 hz ) frequencies were tested . hearing threshold levels were measured in decibels ( db ) . in each case , the threshold level was calculated for both ears and at every frequency . the threshold levels at 250 hz , 500 hz , 1000 hz , and 2000 hz were identified as low - frequency audiometry , the thresholds at 4000 hz , 6000 hz , and 8000 hz as high - frequency audiometry and the thresholds at 9000 hz , 10000 hz , 11200 hz , 12500 hz , 14000 hz , 16000 hz , 18000 hz , and 20000 hz as extended - high ( eh ) frequency audiometry and the mean value was calculated . the mean hearing thresholds of the pcos group and control group at pta - low , pta - high , and pta - eh frequencies were compared . the physical examination of each of the women was performed by the same endocrinology specialist . bmi was calculated by dividing the weight by the square of height ( kg / m ) . the degree of hirsutism was determined using the ferriman - gallwey scoring ( fgs ) system . blood samples were obtained during the follicular phase of the menstrual cycle after 12 hours fasting . the fasting plasma glucose levels ( fpg ) were evaluated using roche hitachi pp modular analyzer ( roche - hitachi , tokyo , japan ) by enzymatic colorimetric assay . follicle - stimulating hormone ( fsh ) , luteinizing hormone ( lh ) , total testosterone ( tt ) , sex hormone - binding globulin ( shbg ) , and insulin measurements were done using the chemiluminescent immunoassay method with an immulite 2000 ( diagnostic products , los angeles , ca , usa ) analyzer . insulin resistance was measured using the homa - ir ( homeostasis model assessment for insulin resistance ) index . the free testosterone index ( fti ) was calculated according to the formula 100 serum testosterone ( nmol / l)/shbg ( nmol / l ) . the descriptive statistics of the variables studied were given as mean , standard deviation , and minimum and maximum values . the student t - test was used to compare data between the control group and pcos group . the statistical significance level was taken as 5% , and spss ( version 21 ) was utilized for all statistical calculations . the demographic data and laboratory findings of the study groups are presented in table 1 . significantly higher fgs , fpg , insulin , homa - ir , tt , fti , and lh / fsh measurements were identified in the pcos group than in the control group ( p=0.001 , 0.009 , 0.013 , 0.005 , 0.001 , 0.001 , and 0.003 , respectively ) . the comparison of the low , high , and eh frequency hearing thresholds of the pcos and control groups is presented in table 2 . there was no statistically significant difference between the mean hearing thresholds of the groups at low or high frequencies . on the other hand , the mean hearing thresholds at eh frequencies were statistically significantly higher in the pcos group than in the control group ( p=0.001 for the right ear and p=0.015 for the left ear ) . the correlation analysis of the pcos group showed a positive correlation between the eh frequency hearing threshold levels and the fti values ( r=0.380 , p=0.016 for the right ear and r=0.360 , p=0.022 for the left ear ; figure 1a , 1b ) . also , a significant positive correlation was identified between the hearing thresholds at eh frequencies and the fgs value ( r=0.344 , p=0.030 for the right ear and r=0.432 , p=0.005 for the left ear ; figure 2a , 2b ) . but there was no significant correlation between the eh frequency and bmi , fpg , insulin , homa - ir , lh / fsh ( p>0.005 ) in the pcos group . comparison of the pcos and control groups in terms of the average hearing thresholds at each frequency in pta - eh measurements are shown in table 3 . hearing thresholds at each frequency in pta - eh were statistically significantly higher in the pcos group than in the control group except for left ear 20000 hz . there are various previous studies reporting that pcos and hearing loss are associated at high ( 40008000 hz ) and eh ( 800014000 hz ) frequencies . these studies do not provide any information about the presence of a relationship between hearing loss and known parameters of pcos such as ir , elevated lh / fsh ratio , hirsutism score , and elevated fti . to our knowledge , our study is the first study to investigate the association of hearing thresholds with parameters such as ir , lh / fsh ratio , hirsutism score , and fti in pcos cases . in histological examination , hwang et al . detected the presence of microvascular structures with leaner walls in the stria vascularis of the basal end of the cochlea in obese rats . they also reported that markers such as tumor necrosis factor alpha , hypoxia - induced factor 1 , caspase 3 , poly ( adp - ribose ) polymerase-1 , nuclear factor kappa b , and apoptosis inducing factor are present in the basal end of the cochlea at high concentrations . various methods have been used to measure hearing thresholds . in our study , the hearing threshold levels at eh frequencies ( 900020000 hz ) were statistically significantly higher in the pcos group when compared to the control group . however , at low ( 2502000 hz ) and high ( 40008000 hz ) frequencies , there was no significant difference between the hearing thresholds of the pcos group and the control group . our findings support other studies that also reported findings suggesting that eh frequencies which represent the basal end of the cochlea are often influenced sooner in disorders that cause endothelial and vascular damage . in one other study , it was reported that chronic inflammation in pcos could induce of hearing loss , particularly at eh frequencies . as exposure to the multifactorial agents of pcos continues over time , it was determined that hyperandrogenism was associated with elevated blood pressure , low hdl cholesterol , dysglycemia , insulin resistance , and abdominal obesity [ 2227 ] . endothelial damage and increased risk for cardiovascular disease in pcos patients have also been reported . some of the biochemical and hormonal changes that occur in pcos have been identified as responsible for atherosclerosis and endothelial dysfunction . in light of all this information , it can be suggested that the hyperandrogenism in pcos impairs microcirculation especially in the basal end of the cochlea through microangiopathy and leads to an elevation in hearing thresholds at eh frequencies . in our study , we detected the presence of a positive correlation between the fti and fgs values and hearing thresholds at eh frequencies in the pcos group . however , there was no significant correlation between bmi , fpg levels , insulin levels , ir , or lh / fsh ratio and hearing thresholds at eh frequencies . these results support the view that the elevation of testosterone levels ( the fundamental cause of hirsutism in pcos ) may play a significant role in the pathophysiology of elevated hearing thresholds . first , it is a cross - sectional study and therefore is incapable of assessing the changes that may occur in hearing levels over time ; new longitudinal studies are required to investigate the causal role of pcos in hearing loss . new studies conducted with larger case populations are needed to elucidate the influence of pcos and various risk factors on auditory functions . at pta - eh frequencies , we detected significantly higher hearing thresholds in pcos patients than in controls . we also determined that elevated fti and fpg levels were positively correlated with elevated hearing thresholds . these findings support the position that one of the most important factors playing a role in the elevation of hearing thresholds in pcos is hyperandrogenism , which is a fundamental component of pcos . a positive correlation was identified between elevated hearing thresholds and hirsutism scores ( fgss ) , which can easily be calculated during physical examination . we believe that pcos - related hyperandrogenism plays an important role in sensorineural hearing loss and that this effect can be avoided by managing identified risk factors . also , early hearing screening may help diagnose hl early , especially in pcos cases with significant hyperandrogenism and hirsutism .

we describe a case of takotsubo cardiomyopathy in an elderly woman after status epilepticus . in an emergency echocardiography , not only left ventricular apical ballooning but also right ventricular apical hypokinesia was observed . after a medical management , the patient 's condition was improved and a follow - up echocardiography showed substantial recovery of left and right ventricular apical ballooning . backgroundthe purpose of our study was to investigate the association between polycystic ovary syndrome ( pcos ) and hearing thresholds.material/methodsforty women diagnosed with pcos ( mean age , 24.336.38 years ) and 40 healthy women controls ( mean age , 26.386.75 years ) were included in prospective study . each case was tested with low ( 250 , 500 , 1000 , and 2000 hz ) , high ( 4000 , 6000 , and 8000 hz ) and extended high ( eh ) ( 900020000 hz ) frequency audiometry . the fasting plasma glucose , insulin , fsh , lh , total testosterone , and sex hormone - binding globulin were measured in all patients.resultsthe mean hearing thresholds at eh frequencies were statistically significantly higher in the pcos group than in the control group ( p=0.001 right ear and p=0.015 left ear ) . there were significant positive correlations among free testosterone index ( fti ) values and hirsutism scores with eh frequency hearing thresholds.conclusionsat pure - tone audiometry ( pta ) eh frequencies , we detected significantly higher hearing thresholds in pcos patients than in controls . we also determined that elevated fti and hirsutism score were positively correlated with elevated hearing thresholds in eh frequencies . these findings support that hyperandrogenism can play a role in the elevation of hearing thresholds in pcos .

retinal artery macroaneurysms ( rams ) are rare , acquired dilatations of the retinal arterial vasculature , usually within the first three branches of the arteriolar tree.1 the incidence is estimated to be approximately 1 in 4,500 people over the age of 40.2 hypertension and older age lead to hyaline degeneration of the vascular walls , loss of autoregulatory tone and elastic recoil , and arterial dilatation.3 approximately 8%25% of rams will undergo spontaneous involution . even with submacular hemorrhage , up to 37% of patients will have better than 20/40 visual acuity without treatment.4 however one third of patients will develop vascular leakage and retinal edema and may need treatment.5 an 84-year - old female with a past medical history of hypertension and dyslipidemia was referred for progressively decreasing visual acuity in the left eye . she had no history of diabetes . on initial examination , dilated fundus examination and fluorescein angiography of the left eye revealed a superotemporal retinal arterial macroaneurysm , with surrounding circinate exudates involving the fovea ( figures 1 and 2 ) . the patient was observed initially , but the edema increased and her vision deteriorated to 20/60 . the patient was observed for 3 months , but the vision worsened again to 20/60 , with increased edema and exudates on optical coherence tomography . she was observed for 3 months , but due to recurrent leakage , she ultimately received a total of six injections of intravitreal bevacizumab every 1 month to 3 months . argon laser at a setting of 400 mw and 200 m spot size was applied around the ram . the ram occluded , and on follow - up examination 6 months later , the patient s vision has remained at 20/30 . the first report of laser photocoagulation to treat exudative retinal artery macroaneurysm was published in 1976 by dr donald gass and colleagues.6 in a study of 27 patients with symptomatic ram , half of the patients underwent argon laser therapy and the remaining half was observed . the vision in both groups improved , but the laser group experienced a greater gain.7 approximately 16%27% of rams occlude with lasers , but there is a risk of vascular occlusion , early increase in exudates from selective reabsorption of fluid , arteriovenous shunts , macular pucker , and scotomas.7,8 other treatment options for rams include yellow dye laser and indocyanine green dye - enhanced photocoagulation.9,10 anti - vascular endothelial growth factor ( vegf ) therapy has recently been reported in the treatment of patients with exudative or hemorrhagic rams . chanana and azad11 published the first case report in 2009 , and subsequent case reports have shown encouraging results ( table 1).1219 cho et al18 described 23 patients with rams who were either observed or received intravitreal bevacizumab . both groups experienced statistically significant improvements in visual acuity and central macular thickness , but the bevacizumab group regained vision faster . a larger , prospective study of 38 eyes with hemorrhagic and exudative rams underwent three monthly injections of bevacizumab , with the vision and retinal thickness improving in both groups.19 the role of vegf and the mechanism of action of anti - vegf therapy in rams are not fully understood . in a study that compared the vegf levels in 500 l vitreous samples from patients with vitreous hemorrhage from nondiabetic etiologies ( including four patients with ram ) with those from patients with proliferative diabetic retinopathy , the vegf levels were significantly lower in the former than in the latter groups ( 2.75 pg / ml vs 821 pg / ml , respectively).20 however , these were small vitreous samples obtained up to 3 weeks after the initial hemorrhage and may not have been representative of the vitreous and microenvironment around the ram . the role of vegf in intracranial aneurysms and other biological systems has been more clearly elucidated . in intracranial aneurysms , vegf levels were found to be significantly higher than the levels in controls.20 in particular , patients with intracranial aneurysms had higher expressions of vegf receptor 2 ( vegfr2 ) and lower levels of vegf receptor 1 ( vegfr1 ) . vegfr1 is associated with angiogenesis , while vegfr2 is associated with thrombosis.21 bevacizumab has been shown to decrease vegfr1 levels and to increase vegfr2 levels to normal levels in cancer cells.22 in the circulatory system , platelets produce vegf , which stimulates nitrous oxide production , resulting in vasodilation.21 inhibition of vegf may decrease leakage of fluid and exudates through the endothelium . in mice studies , bevacizumab decreases bleeding time and increases coagulation.23 thus , vegf inhibition may block angiogenesis , decrease binding of the prothrombotic vegfr2 , and decrease vascular permeability . in conclusion , the majority of rams can be observed , but intervention may be necessary in cases of persistent or recurrent macular edema from exudative or hemorrhagic rams . our patient s ram was initially observed , but continued leakage prompted further therapy . anti - vegf injections have shown promise in improving visual acuity and hastening resolution of macular edema in ram;5 however , most of the previously published cases had relatively short follow - up . our case has one of the longest follow - ups with the most number of anti - vegf treatments , but the patient continued to have leakage into the macula . the potential benefits of anti - vegf treatment must be carefully weighed against the potential risk of infection from serial injections . laser photocoagulation remains a viable treatment option in cases of macular edema secondary to ram . the absorption of iron is strictly controlled through hepcidin , while there are no efficient physiologic mechanisms to excrete iron from the body . under conditions where iron metabolism is disturbed , such as in transfusion - dependent patients with thalassemia major , myelodysplastic syndromes , aplastic anemia , or sickle cell disease , excessive iron deposition occurs in the liver , heart , and endocrine organs , resulting in reactive oxygen species ( ros ) through the fenton and haber weiss reaction . ros can attack components of cells and further induce organ dysfunction , such as liver fibrosis , cirrhosis , hepatic carcinoma , myocarditis , pericarditis , and other diseases . in the liver of chronic hepatitis c ( chc ) patients , iron deposition has been found in both hepatocytes and reticuloendothelial cells although the mechanism is not fully clarified . like other viruses , hepatitis c virus ( hcv ) needs constituents of host cells to proliferate , and iron is one of the most important constituents . many studies have explored the connection between iron overload and hcv life cycle with differing results . some studies have found that iron promotes hcv replication , while others have shown that iron suppresses hcv replication . most of the studies suggest the positive role of iron on hcv translation . to summarize the interactions between hcv and iron , and understand the mechanisms of iron overload in chc , we searched articles published in the pubmed databases up to january 28 , 2017 , to gain an in - depth understanding and knowledge of relevant areas . in the human body , iron is critical for maintaining the fundamental function of many proteins . iron in the diet is absorbed through divalent metal transporter 1 ( dmt1 ) , a multi - transmembrane protein , or heme carrier protein 1 on duodenal and jejunal enterocytes . it is then exported by the ferroportin ( fpn ) to bind to transferrin in the bloodstream , and taken to the erythroblasts for erythropoiesis . iron that is not utilized can also be stored as ferritin or hemosiderin in enterocytes , macrophages , and hepatocytes . there are no efficient physiologic mechanisms to excrete iron , a little iron ( about 2 mg per day ) is lost by sloughing of intestinal epithelial cells , desquamation of skin and urinary cells , blood loss , and sweat . iron homeostasis in the human body is mainly regulated by the hepcidin / fpn and iron - regulatory protein / iron - responsive element ( irp / ire ) systems . when iron levels increase , hepcidin negatively regulates iron levels by binding to the fpn and promoting the internalization and degradation of fpn . this reduces the amount of iron absorbed by enterocytes , released by hepatocytes , and recycled from macrophages , and finally reduces transferrin levels in the bloodstream . in contrast , when iron levels decrease , hepcidin is downregulated to improve transferrin levels . transferrin receptor 1 ( tfr1 ) and bone morphogenetic protein 6 receptor ( bmp-6r ) can sense transferrin saturation ( ts ) and tissue iron content , respectively , further regulating hepcidin concentration . hepcidin can also be affected by other factors , including inflammation , erythropoietic activity of the bone marrow , and the oxygen tension within hepatocytes . while hepcidin regulates systemic iron metabolism transcriptionally and posttranslationally , cellular iron metabolism is regulated by the irp / ire system posttranscriptionally . the irp / ire system alters the expression of iron - metabolism - related proteins , such as ferritin , fpn , hypoxia - inducible factor 2 ( hif2/epas1 ) , dmt1 , tfr1 , and other proteins . many studies have found iron deposition in the liver , and in both hepatocytes and reticuloendothelial cells . were the first to find elevated serum ferritin and iron levels in patients with chronic hepatitis ( both chronic hepatitis b and chc ) . subsequently , in 1999 , a study of 209 chc patients found liver iron accumulation detected by liver biopsy in 42.1% of patients , the majority of which was mild liver iron accumulation ( 35.4% of the 209 chc patients ) . those with liver iron accumulation had significantly higher levels of serum iron ( si ) , ferritin , and ts ; liver iron accumulation was also found to have a significant relationship with the severity of histological activity based on metavir classification and cirrhosis . in a study of 100 consecutive patients with hcv infection who underwent liver biopsy , 19 patients were found to be hepatic iron stain positive , which is associated with stage iii or iv fibrosis . fifty - five patients had at least one abnormal value of si , ferritin , or ts . in multivariate analysis , the only independent predictive factor of severe hepatic fibrosis was serum ferritin . however , elevated serum ferritin can also be caused by hepatic inflammation . in 1994 , a study of 123 chronic hepatitis patients ( including 63 chc patients ) found increased si , iron saturation , and ferritin in chc patients , while no evidence of hepatic iron accumulation could be found in any of these patients . in this study , serum ferritin was elevated in the absence of liver iron overload , which indicates that elevated serum ferritin may also be caused by inflammation itself . thus , elevated ferritin reflects hepatic iron accumulation , as well as hepatic inflammation , and also predicts severe hepatic fibrosis . in other words , hepatic iron accumulation can lead to elevated ferritin , while elevated ferritin is not only induced by hepatic iron accumulation but also caused by hepatic inflammation . thus , serum ferritin levels only serve as a reference to evaluate liver iron status in chc patients , and liver biopsy is the gold standard for diagnosis of iron overload . so far , plenty of studies have been devoted to exploring the mechanism as to how hcv leads to iron overload . it is generally accepted that hcv alters iron metabolism by reducing the level of hepcidin . fujita et al . measured hepcidin messenger rna ( mrna ) levels in liver samples from 56 patients with hcv infection , and revealed that the expression of hepcidin is strongly correlated with serum ferritin and the degree of iron deposition in liver tissues . the hepcidin - to - ferritin ratio was significantly lower in hcv(+ ) patients than hbv(+ ) patients or controls . girelli et al . detected s - hepcidin ( the 25-amino acid bioactive peptide in serum ) levels in 81 untreated chc patients and 57 controls with a rigorous definition of normal iron status and found that s - hepcidin was significantly lower in chc patients than in controls . in chc patients , s - hepcidin levels examined the determinants of serum hepcidin and liver hepcidin mrna levels and their association with histological lesions in 96 patients with chc and 30 controls ; they concluded that serum hepcidin was significantly lower in patients with chc compared to healthy controls , and that liver hepcidin mrna levels did not differ between patients and controls . a study using fl - n/35 transgenic mice harboring the hcv polyprotein also found decreased hepcidin expression in the liver , accompanied by an increase in fpn expression in the duodenum , spleen , and liver . this suggests that hcv proteins may directly lead to increased duodenal iron absorption , macrophage iron release , and hepatic iron accumulation . elevated duodenal fpn levels and a significant relationship between hcv and hepcidin in chc patients were also found by other studies . all of these studies suggest that hepcidin may play a pivotal role in the pathogenesis of iron overload in patients with chc . kohjima et al . analyzed iron - metabolism - related gene expression profiles in 100 patients with chc ( genotype 1b , n = 50 ; genotype 2 , n = 50 ) and 18 living donors of liver transplantation . they found that expression of genes related to iron absorption ( transferrin , and dmt1 ) , iron export ( fpn ) , cellular iron metabolism ( irp1 and irp2 ) , and hepcidin - regulation ( bmpr1 , bmpr2 , and hemojuvelin ) were significantly higher , indicating that hcv affects the expression of iron - metabolism - related genes , leading to iron accumulation in hepatocytes . besides , patients with a sustained virological response ( svr ) had significantly lower transcription and protein expression levels of hepcidin , fpn , bmpr2 , and hemojuvelin before therapy , indicating the importance of hepatocytic iron retention for viral response during pegylated interferon plus ribavirin treatment . almost all hcv proteins , including core , e1 , e2 , ns3 , ns4a , ns4b , and ns5a , have been shown to regulate hepcidin expression through signal transducer and activator of transcription 3 , mitogen - activated protein kinase , or bmp / smad signaling pathways , and increased histone deacetylase activity , which further clarifies the mechanism of decreased levels of hepcidin caused by hcv . like other viruses , hcv needs constituents of host cells to replicate and translate , and iron is one of the most important constituents . it is indispensable for several basic metabolic processes in viruses , as well as in mammalian cells , as described in many studies . were the first to analyze the effect of iron on hcv replication in vitro . in 2000 , they cultured a nonneoplastic hcv - infected human hepatocyte line ( ph5ch8 ) treated with feso4 and proved that iron can promote hcv replication in liver cells . in 2016 , foka et al . found that iron - overloaded macrophages was infected with hcv when co - cultured with hcv - infected human hepatoma cell line ( huh7.5 ) . these iron overloaded macrophages also enhanced hcv replication in co - cultured hcv - infected huh7.5 cells through reversed ferritin flow from macrophages to huh7.5 cells . macrophages without overloaded iron was also infected with hcv and enhance hcv replication in huh7.5 cells , but the rate of infection was slower and the effect on hcv replication was weaker . on the other hand , several studies have found that iron suppresses hcv replication . a study using huh7 cells in 2005 concluded that iron could inhibit hcv replication by inactivating the rna polymerase ns5b , without significant effect on translation . fillebeen et al . concluded that iron could decrease hcv replication by reducing the activity of ns5b in huh7.5.1 cells , similar to the result of the study in 2005 . a later study , in 2011 , again found that increased iron status and down - regulated hepcidin inhibited hcv replication . . this may be due to different cell types . besides , in clinical practice , chc with primary or secondary iron overload have different prognoses . chc patients with hereditary iron overload due to hemochromatosis gene mutations have increased svrs to antiviral therapy , when excess iron deposits in parenchymal cells and macrophages become iron deficient . in contrast , iron overload secondary to chc or other chronic liver diseases significantly aggravates the disease , when excess iron deposits in macrophages . it seems that the iron content of macrophages is associated with the clinical outcome of disease , because iron overload may impair the immune function of macrophages through oxidative stress . although the role of iron in promoting or suppressing hcv replication is debatable , there are few controversies about the effect of iron on hcv translation . apart from a group in the usa that reported that iron suppresses hcv translation through increasing heme oxygenase-1 in vitro and another group that found no effect of iron on hcv translation , all other studies confirm the positive role of iron on hcv translation , but the mechanisms are not fully understood . expression of hcv is predominantly controlled at the translational level , which involves the interaction between cellular translation initiation factors and a specific mrna stem - loop structure the internal ribosome entry site ( ires ) within the 5 untranslated region . iron is reported to stimulate the expression of eukaryotic initiation factor 3 ( eif3 ) , thus enhancing hcv ires - dependent translation both in hepg2 cells and hcv - infected patients , whereas iron chelation reverses it in vitro . another group also found that hepatic iron load promotes hcv translation initiation in vitro with the mechanism involving iron - dependent increased expression of eif3 . expression of another element , la protein , is also significantly increased by hepatic iron load which can promote hcv translation . expression of both eif3 and la protein can be partially inhibited by the iron chelator deferoxamine . cho et al . also found that iron changes the affinities of common cellular factors to hcv ires which modulates hcv ires - dependent translation . in summary , iron can regulate hcv ires - dependent translation by increasing expression levels of associated factors . translation of both hcv 1b and hcv 2b is significantly increased after iron treatment , while translation of hcv 6a shows little difference . compared with subtype 1b , hcv 6a has a modified eif3 binding site within hairpin iii . it is reported that hepatic iron levels are higher in patients infected with subtype 1b than those in patients infected with subtypes 2a or 2b , which may explain the different responses of hcv genotypes to iron overload . nowadays , liver biopsy is the gold standard for diagnosis of hepatic iron overload , and there are no uniform diagnostic criteria about extracellular iron concentration . most of the studies described in the paper used cell models incubated in ferric or ferrous iron solutions with maximum concentrations ranging from 50 to 300 mol / l , whether they reach the standard of iron overload is difficult to judge . therefore , whether these results from in vitro research could be suggestive to understand the effect of iron on hcv is still debatable . each part of iron metabolism , including absorption , restoration , recycling , and utilization , is regulated by hepcidin and the ire / irp system . there is no efficient way to eliminate iron from the body , so any abnormality in the process of iron metabolism may lead to excessive iron burden . the relationship between hcv and iron overload many studies have observed iron overload in patients with chc , and most suggest that decreased hepcidin expression , as well as increased hcv protein - mediated expression of other iron metabolism - related genes , play a role . as to the detailed mechanism of hcv proteins inducing iron overload , there is still much to discover . in turn , as an essential element for all living bodies , iron can also affect the life cycle of hcv . nearly all studies so far confirm the positive role of iron on hcv translation , while the role of iron in promoting or suppressing hcv replication is still unclear . more work is needed to understand this , and the mechanisms by which iron affects hcv life cycle . iron chelation therapy can reverse the negative effect of iron overload , which offers important information for clinical application . hcv reduces the levels of hepcidin , and elevates the levels of fpn , resulting in elevated iron absorbed by enterocytes and iron deposition in hepatocytes and macrophages . although the role of iron in hcv replication is disputable , it is strongly suggested the positive effect of iron on hcv translation by increasing expression of both eif3 and la protein , and changing the affinities of common cellular factors to hcv ires . hcv : hepatitis c virus ; fpn : ferroportin ; si : serum iron ; ts : transferrin saturation ; ires : internal ribosome entry site ; eif3 : eukaryotic initiation factor 3 ; : upregulated ; : downregulated . this work was supported by grants from the 215 project of beijing municipal commission of health and family planning ( no . 303 - 01 - 005 - 0068 ) and the basic - clinic cooperative foundation of capital medical university ( no . this work was supported by grants from the 215 project of beijing municipal commission of health and family planning ( no . 303 - 01 - 005 - 0068 ) and the basic - clinic cooperative foundation of capital medical university ( no .

an 84-year - old female with a history of hypertension and dyslipidemia was referred for a retinal artery macroaneurysm with exudation that had extended into the macula . she underwent a total of six injections of bevacizumab , with some improvement in visual acuity and retinal thickness . due to persistent macular edema , focal laser photocoagulation was performed around the macroaneurysm . the vision remained at 20/30 during 20 months of follow up . although anti - vascular endothelial growth factor therapy may improve vision and decrease retinal thickness in retinal artery macroaneurysm , recalcitrant cases may be treated with laser photocoagulation to seal the leaking vessel . objective : the aim of this study was to summarize the interactions between hepatitis c virus ( hcv ) infection and iron overload , and to understand the mechanisms of iron overload in chronic hepatitis c ( chc ) and the role iron plays in hcv life cycle.data sources : this review was based on data in articles published in the pubmed databases up to january 28 , 2017 , with the keywords hepatitis c virus , iron overload , iron metabolism , hepcidin , translation , and replication.study selection : articles related to iron metabolism , iron overload in patients with chc , or the effects of iron on hcv life cycle were selected for the review.results:iron overload is common in patients with chc . the mechanisms involve decreased hepcidin levels caused by hcv through signal transducer and activator of transcription 3 , mitogen - activated protein kinase , or bone morphogenetic protein / smad signaling pathways , and the altered expression of other iron - metabolism - related genes . some studies found that iron increases hcv replication , while other studies found the opposite result . most of the studies suggest the positive role of iron on hcv translation , the mechanisms of which involve increased expression levels of factors associated with hcv internal ribosome entry site - dependent translation , such as eukaryotic initiation factor 3 and la protein.conclusion:the growing literature demonstrates that chc leads to iron overload , and iron affects the hcv life cycle in turn . further research should be conducted to clarify the mechanism involved in the complicated interaction between iron and hcv .

colorectal cancer ( crc ) is a major cause of mortality and morbidity , and the third most common malignancy in the world ( 1 ) . the incidence of this malignancy shows considerable variation among racially or ethnically defined populations in multiracial / ethnic countries . crc is the third most common type of cancer in men and the second most common cancer in women worldwide ( 2 ) . kashmir has been reported as being a high - incidence area of gastrointestinal ( git ) cancers ( 3,4 ) . in the kashmir valley , crc represents the third most common type of git cancer , following esophageal and gastric cancer ( 5,6 ) . the tumour - suppressor gene p16ink4a codes for a cyclin - dependent kinase inhibitor p16ink4a , which acts as a negative regulator of cell growth and proliferation in the g1 phase of the cell cycle ( 7,8 ) . functionally , p16ink4a has been identified as an inhibitor of cyclin - dependent kinase 4 and 6 . as a consequence , p16ink4a plays a significant role in the proliferation process of many types of tumour cell growth and apoptosis , and correlates closely with the expression of the proliferating cell nuclear antigen ki67 , which is usually viewed as the proliferation marker in intestinal tumours ( 9,10 ) . most human colorectal carcinomas show genetic alterations in the p16ink4a - cyclin d - prb pathway ( 11 ) . the p16ink4a gene of the cyclin d / prb pathway has been found to be inactivated in human malignancies with a frequency second only to p53 ( 12,13 ) . mutation , homozygous deletions and hypermethylation of the promoter are major mechanisms of p16ink4a inactivation ( 8,14,15 ) . hypermethylation of non - mutated promoter regions is one of the common mechanisms for inactivating tumour - suppressor genes , which leads to stable allele - specific loss of transcription function ( 16 ) . such methylations tend to occur at the sites of cpg dinucleotides , which are clustered as so - called cpg islands and are frequently found in promoters of p16ink4a and other tumour - suppressor genes ( 13 ) . in fact , the major mechanism of p16ink4a gene inactivation has been found to be promoter methylation ( 11 ) . a number of studies have been carried out on p16ink4a in different populations , implicating the role of hypermethylation in the development of cancers ( 1723 ) . p16ink4a gene promoter methylation has been observed in colorectal dysplasia , adenomas , malignant tumours and normal mucosa adjacent to tumours ( 18,19,24 ) . two significant investigations were previously carried out in the kashmir valley in order to establish the role of p16ink4a mutations and promoter hypermethylation in gastric and esophageal squamous cell carcinoma , respectively ( 13,25 ) . based on the hypothesis that crc carcinogenesis is a multi - step and multi - gene event , we designed this study to elucidate the role of p16ink4a promoter hypermethylation in the development and progression of crc in the kashmiri population and to correlate it with the clinicopathological parameters of crc cases . this study included 114 crc cases recruited from the department of surgery , sher - i - kashmir institute of medical sciences ( skims ) , srinagar , india . tumour and adjacent normal tissue samples from the cases were resected in the general surgery department ( skims ) and were collected for this study . data on all crc cases were obtained from personal interviews with patients and/or guardians and medical records . all patients and/or guardians were informed of the study and their will to participate in this study was noted in a pre - designed questionnaire ( available on request ) . samples were snap - frozen immediately after collection and stored at 70c until further analysis . dna was then isolated from both tissue and blood samples using the ammonium acetate method succeeding proteinase - k digestion . the tissue for dna extraction from the tumour sample was selected by an experienced pathologist and was ascertained to comprise > 90% tumour cells . both normal and tumour dnas were subjected to sodium bisulphite modification using the ez dna methylation kit ( zymo research , usa ) . previously reported primer sets were used for the amplification of the p16 promoter ( 11,26 ) . the unmethylated primer pair p16-u1f / u1r produced an amplicon of 151 bp , while p16-u1f / u2r produced an amplicon 234 bp in length . / m1r produced an amplicon of 150 bp , while p16-m1f / m2r produced an amplicon of 234 bp in length . the presence of unmethylation and/or methylation of the p16 promoter was ascertained by the presence of both amplicons . pcr for both unmethylation as well as methylation detection was performed in a 50-l total volume reaction mixture containing 10 ng of modified genomic dna , 100 m of each dntp , 100 ng of each of the three primers ( u1f , u1r and u2r in the case of unmethylation detection ; m1f , m1r and m2r in the case of methylation detection ) , 1.5 mm mgcl2 , 5% dimethyl sulphoxide ( dmso ) , 10x taq buffer and 2 units taq dna polymerase ( fermentas , md , usa ) . the conditions of pcr were as follows : initial denaturation at 95c for 7 min , 40 cycles of denaturation at 95c for 30 sec , annealing at designated temperatures ( c ) ( see table i ) for 45 sec and extension at 72c for 45 sec , and final extension at 72c for 10 min in a bio - rad icycler . the pcr amplicons were electrophoresed on 2.5% agarose gels and visualised after staining with ethidium bromide . universal methylated human dna ( zymo research ) was used as a positive control for methylated alleles whereas dna from normal lymphocytes was used as a control for unmethylated alleles . all statistical analyses were performed using pasw software , version 18 ( ibm , ny , usa ) . pearson s chi - square two - proportion test was used to evaluate the hypothesis of equal distribution of molecular alterations with different clinicopathological variables . a fisher s two - tailed test ( p - values ) of 0.05 was considered statistically significant . this study included 114 crc cases recruited from the department of surgery , sher - i - kashmir institute of medical sciences ( skims ) , srinagar , india . tumour and adjacent normal tissue samples from the cases were resected in the general surgery department ( skims ) and were collected for this study . data on all crc cases were obtained from personal interviews with patients and/or guardians and medical records . all patients and/or guardians were informed of the study and their will to participate in this study was noted in a pre - designed questionnaire ( available on request ) . samples were snap - frozen immediately after collection and stored at 70c until further analysis . dna was then isolated from both tissue and blood samples using the ammonium acetate method succeeding proteinase - k digestion . the tissue for dna extraction from the tumour sample was selected by an experienced pathologist and was ascertained to comprise > 90% tumour cells . both normal and tumour dnas were subjected to sodium bisulphite modification using the ez dna methylation kit ( zymo research , usa ) . previously reported primer sets were used for the amplification of the p16 promoter ( 11,26 ) . the unmethylated primer pair p16-u1f / u1r produced an amplicon of 151 bp , while p16-u1f / u2r produced an amplicon 234 bp in length . / m1r produced an amplicon of 150 bp , while p16-m1f / m2r produced an amplicon of 234 bp in length . the presence of unmethylation and/or methylation of the p16 promoter was ascertained by the presence of both amplicons . pcr for both unmethylation as well as methylation detection was performed in a 50-l total volume reaction mixture containing 10 ng of modified genomic dna , 100 m of each dntp , 100 ng of each of the three primers ( u1f , u1r and u2r in the case of unmethylation detection ; m1f , m1r and m2r in the case of methylation detection ) , 1.5 mm mgcl2 , 5% dimethyl sulphoxide ( dmso ) , 10x taq buffer and 2 units taq dna polymerase ( fermentas , md , usa ) . the conditions of pcr were as follows : initial denaturation at 95c for 7 min , 40 cycles of denaturation at 95c for 30 sec , annealing at designated temperatures ( c ) ( see table i ) for 45 sec and extension at 72c for 45 sec , and final extension at 72c for 10 min in a bio - rad icycler . the pcr amplicons were electrophoresed on 2.5% agarose gels and visualised after staining with ethidium bromide . universal methylated human dna ( zymo research ) was used as a positive control for methylated alleles whereas dna from normal lymphocytes was used as a control for unmethylated alleles . all statistical analyses were performed using pasw software , version 18 ( ibm , ny , usa ) . pearson s chi - square two - proportion test was used to evaluate the hypothesis of equal distribution of molecular alterations with different clinicopathological variables . a fisher s two - tailed test ( p - values ) of 0.05 was considered statistically significant . the patients comprised 67 males and 47 females ( male / female ratio of 1.42 ) . the demographic and clinical characteristics of the crc cases are shown in table ii . out of 114 confirmed cases of crc , 109 cases were sporadic , 4 were familial adenomatous polyposis ( fap ) and 1 was lynch syndrome . all but 1 case were adenocarcinoma and only 1 was squamous cell carcinoma ( scc ) of basal cell type ; 79 came from a rural and 35 from an urban environment ; 49 cases had carcinoma in the colon and 65 in the rectum ; 74 were smokers and 40 non - smokers ; and 89 cases had well - differentiated adenocarcinoma . ms - pcr analysis revealed a high methylation status of the p16ink4a promoter in crc cases . forty - eight ( 42.1% ) of the tumours were found to be hypermethylated at the promoter region of p16ink4a , while 8 ( 7% ) of the tumours were partially hypermethylated and the remaining 58 ( 50.9% ) of the tumours were not methylated at all ( table ii ) . among the 48 patients with tumours which were methylated for the p16ink4a gene promoter , 26 were female , 36 had a higher tumour stage ( c+d ) , 38 were smokers and 31 were of a well - differentiated ( wd ) grade ( table ii ) . statistical analysis between the p16ink4a hypermethylation status and clinicopathological parameters of the crc cases revealed a significant association ( p<0.05 ) with gender ( females ) , lymph node status ( present ) , tumour stage ( c+d ) , smoking status ( smokers ) and tumour grade ( wd ) of the crc patients ( table ii ) . this is the first study to report on the association of p16 gene promoter hypermethylation with the risk of development of crc in the kashmiri population . the kashmir valley , located in the northern part of india and walled by the himalayas has a unique ethnic population living in discrete temperate environmental conditions and having unique eating habits , which , along with genetic factors , play a large role in the development of git cancers ( 46,13 ) . as previously reported , the etiology and incidence of various git cancers in this population has been attributed to a probable exposure to nitroso compounds , amines and nitrates reported to be present in local foodstuffs , such as hoakhe suen ( sun - dried vegetables ) , pharei and hoggade ( sundried and/or smoked fish and meat ) , hakh ( a leafy vegetable of the brassica family ) , hot noon chai ( salted tea ) , dried and pickled vegetables and red chilli , and also through smoking hukka ( a water pipe ) ( 3,4,27 ) . cancer cell lines have in general demonstrated an increased frequency of hypermethylation in comparison to primary tumours ( 28 ) . the inactivation of tumour - suppressor genes by promoter hypermethylation has been recognised to be as common as gene disruption by mutation in tumourigenesis ( 2931 ) . a number of studies on crc around the globe have demonstrated the role of promoter hypermethylation of a number of different genes in the development and progression of crc ( 3234 ) . promoter hypermethylation of p16ink4a , similarly to that of other genes , plays a pivotal role in the inactivation of p16ink4a , which in turn enhances tumour development ( 11,18,23 ) . in the present study , we observed hypermethylation of the p16ink4a gene promoter in 42.1% ( 48/114 ) of crc cases , which is higher than reported by other major studies in the world ( 11,20,21 ) . however , our observation is consistent with other studies ( 22,23 ) which have reported a similar frequency of p16ink4a hypermethylation in crc tumours in the japanese population . this may be due to the fact that this population is exposed to a special set of environmental challenges , including extreme temperature , high altitude and special food habits , as well as exposure to agricultural by - products , such as pesticides and nitrosamines ( 6,13 ) . liang et al , urosevic et al and krtolica et al have already proposed that geographical differences or other unknown factors supplementary to p16 methylation may increase tumour aggressiveness ( 3537 ) . we found a significant association of the p16ink4a methylation status with higher dukes stage ( c+d ) . these results are in concordance with those of other studies ( 20,21,23 ) , which have reported the same observations of a higher methylation status of p16ink4a gene promoter with higher dukes stage ( c+d ) . duke s staging has been considered a most significant prognostic determinant in cancers ( 20 ) . dukes staging , proposed by dr cuthbert e. duke in 1932 , is a clinical classification for crc based on the tumour size , local extent and metastatic status , i.e. , lymph node involvement . hence , the present study supports the observation of yi et al ( 20 ) in suggesting that p16ink4a methylation may be involved in the malignant transformation of crc . furthermore , a significant association was found between p16ink4a methylation status and lymph node metastasis in our population , as was also reported by goto et al in their study ( 23 ) . we also found that females were more likely than males to have p16ink4a methylated crc tumours in our population ; this supports the study of wiencke et al ( 38 ) . however , contrary to this observation , we found the methylation status of the p16ink4a gene to be associated with a well - differentiated tumour grade . jie et al previously demonstrated the association of p16ink4a methylation status with the proliferative activity of crc ( 39 ) . they reported the loss of proliferation and invasion in crc in tumours with re - expression of p16ink4a protein . thus , epigenetic silencing of p16ink4a may have a role in driving crc tumours to higher differentiation and consequently to invasiveness . another unique observation in our study was the association of the methylation status with smoking status . tumours of the crc patients who had a history of smoking ( cigarettes and/or hookah ) were found to have methylated p16ink4a gene promoter ( p<0.05 ) . in conclusion , in the kashmir valley population , the high level of epigenetic silencing of p16ink4a plays a pivotal role in the initial tumourigenesis and also enhances the chances of tumour development and progression to advanced stages . anabolic androgenic steroids ( aass ) represent a large group of synthetic derivatives of testosterone , produced to maximize anabolic effects and minimize the androgenic ones [ 1 , 2 ] . the most common oral and injectable aass with their chemical structures are reported in table 1 . androgens act by binding to the nuclear androgen receptor ( ar ) in the cytoplasm and then translocate into the nucleus . this binding results in sequential conformational changes of the receptor affecting the interaction between receptor and protein , and receptor and dna . the basic structure of all steroids is a perhydro - cyclopentano phenanthrene ring system that can be modified in order to obtain several designed chemical modifications . the most important chemical modification , in which the basic structure can undergo , is reported in fig . 1 . anabolism is defined by kuhn as any state in which nitrogen is differentially retained in lean body mass , either through stimulation of protein synthesis and/or decreased breakdown of protein anywhere in the body . skeletal muscle can be considered as the main target tissue for the anabolic effects of aas , which are mediated by ars which after exposure to aass are up - regulated and their number increases with body building . therefore , aass determine an increase in muscle size as a consequence of a dose - dependent hypertrophy resulting in an increase of the cross - sectional areas of both type i and type ii muscle fibers and myonucleardomains . moreover , it has been reported that aass can increase tolerance to exercise by making the muscles more capable to overload therefore shielding them from muscle fiber damage and improving the level of protein synthesis during recovery . despite some therapeutic use of aass ( severe burns , primary or secondary hypogonadism , short stature , hiv wasting syndrome etc . ) there is also wide abuse among athletes especially bodybuilders in order to improve their performances and to increase muscle growth and lean body mass , taking into account the significant anabolic effects above reported . not by chance , these substances fall within the vast group of performance - enhancing drugs , which also include : stimulants , painkillers , sedatives and anxiolytics , diuretics , blood boosters and masking drugs . a high - dose regimen is stacked by combining numerous oral and injectable aass , which are self - administered in drug cycles which last from 4 to 12 weeks [ 7 - 9 ] . furthermore , aas users frequently associate other substances to aass , the so called steroid - accessory drugs , such as ephedrine , growth hormone , insulin , diuretics , ghb etc [ 10 - 15 ] for several reasons . the prolonged misuse and abuse of aass can determine several adverse effects , some of which may be even fatal especially on the cardiovascular system because they may increase the risk of sudden cardiac death ( scd ) , myocardial infarction , altered serum lipoproteins , and cardiac hypertrophy . 2 . the aim of this review is to focus on deaths related to aas abuse , trying to evaluate the autoptic , histopathological and toxicological findings in order to investigate the pathophysiological mechanism that underlines this type of death , which is still obscure in several aspects . some databases , from 1975 to june 2014 , were searched : medline , cochrane central , scopus , web of science , science direct , embase and google scholar , using the following keywords : anabolic androgenic steroid , death , cardiovascular effects , toxicity , side / adverse effects . the main key word anabolic androgenic steroid was individually searched in association to each of the others . the 189 sources found after the initial screening in order to exclude duplicate sources and retrospective studies , were selected according to the inclusion criteria , which allowed the identification of 10 sources . a comprehensive flow diagram with inclusion criteria the review of the literature using the flow diagram reported in fig . 3 allowed us to identify 19 fatal cases between 1990 and 2012 . the most important autoptic , histopathological findings and circumstantial data are reported in table 2 , while the toxicological findings are reported in table 3 . of the 19 cases , 17 ( 89.5% ) were males whereas only 2 ( 10.5% ) were females . the age ranged from 18 to 37 years ( mean age : 28 4.4 ) . among the 19 fatal cases , in 14 bodies ( 12 males and 2 females ) the data available allowed to calculate the body mass index ( bmi ) and the results are reported in table 4 . in none of the cases the bmi was lower than 24.9 , which is considered the upper limit of the normal healthy weight . for all cases the autopsy excluded extracardiac causes of death , only in one case a bilateral pulmonary embolism from deep venous thrombus of lower extremities was found ( table 2 ) . toxicological investigation performed mainly on urine samples but also in blood and hair samples , by using several screening tests and analytical methods revealed in 12 cases [ 16 , 18 , 20 - 25 ] the presence of aass and/or their metabolites in urine specimens ; in one case nandrolone was detected in blood , whereas in another case stanozolol was found in hair . in the remaining 6 cases in which the toxicological analysis was negative , circumstantial data and evidences reported by relatives and friends of the deceased highlighted a previous prolonged use of aass . the chronic use of aass can cause various pathological alterations , which are related to dose , frequency and patterns of use . taking into account that numerous organs and apparatus are the target of aass , several adverse effects can involve the liver , cardiovascular , reproductive , musculoskeletal , endocrine , renal , immunologic and hematological systems as well as some psychological effects ; a schematic representation is reported in fig . 19 fatal cases are reported ; although only single case report or small series of cases were included , whereas retrospective studies and other papers that did not fulfill the inclusion criteria were not taken into account , some consideration can be formulated ; in all cases the autopsy findings together with the histological examination have highlighted cardiac causes of death . only in one case a mechanical cardiovascular cause of death was found ( a bilateral pulmonary embolism from deep venous thrombus of lower extremities ) . in numerous cases [ 16 , 18 , 19 , 22 , 25 ] , a common finding was a left ventricular hypertrophy , frequently associated with fibrosis and myocytolisis . a myocardial hypertrophy was not found in the 4 cases reported by fineschi et al in two different reports [ 21 , 23 ] . what is the significance that could be attributed to the myocardial hypertrophy ? a vigorous training in young athletes can determine a left ventricularhypertrophy , independently of the use of aass ( the so called athlete 's heart ) [ 25 - 28 ] . melchert and welder categorized the effects of aas on the cardiovascular system into four groups of activities : vasospastic , atherogenic , thrombotic and direct myocardial injuries . aas can induce adverse cardiovascular effects such as left ventricular hypertrophy ( lvh ) , hypertension , impaired diastolic filling , arrhythmia , erythrocytosis , thrombosis and altered lipoprotein profiles . abnormalities in cardio - vascular reflex control of the cardiovascular system [ 31 - 35 ] and in vascular reactivity [ 36 - 40 ] have also been reported . studies on isolated hearts from rats treated chronically with nandrolonedecanoate ( nd ) have also shown a raise in myocardial susceptibility to ischemia / reperfusion injuries [ 41 , 42 ] . nandrolone abuse combined with vigorous exercise training may lead to impaired diastolic function and concentric hypertrophy of the left ventricular ( lv ) wall . vigorous weight lifting itself would also cause lv wall mass and thickness increase but cardiac function would not be affected . however , when combined with aas abuse pathological cardiac hypertrophy could be caused . in another study , rats were treated with nd for 6 weeks ( total dose 30 mg /kg ) . rocha et al . studied the effects on cardiac function in rats undergoing swimming training and those not undergoing it . they investigated that swimming training combined with high doses of nandrolone ( 5 mg / kg per injection , equal to 10 mg / kg per week ) sharpens cardiac hypertrophy with interstitial fibrosis . without a doubt . an explanation might be the change of the sympathetic autonomic activity modulated by the renin - angiotensin - system ( ras ) . experiments have shown that ras plays a significant role in the development of lvh and myocardial fibrosis . angiotensin ii type 1 receptor s ( at1r s ) stimulation is associated with the regulation of cell growth and proliferation of vascular smooth muscle cells , cardiomyocytes and endothelial cells involved in endothelial dysfunction , atherosclerotic vascular phenomena , congestive heart failure and myocardial infarction . marques neto et al . pointed out that the treatment with supraphysiological , chronic doses of aass induce cardiac parasympathetic disturbances in ventricular depolarization in both exercised and sedentary rats . unambiguously , it has been shown that the blockage of the ras , and in particular of at1r by losartan , obstructs qt prolongation . down - regulation of ion channel subunits , kchip2 , kv1.4 and kv4.3 , could explain the autonomic dysfunction and cardiac repolarization disturbances caused by chronic treatment with supraphysiological doses of nd . moreover , prolonged qt intervals and ventricular action potential could be explained by the reduced density of the transient outward potassium . no augmentation in tissue collagen content or in the mrna expression of types i and iii collagens have been shown by histological analysis the previously mentioned unconformity could be attributed to the duration of treatment with nd and the age of rats used . participation of the potassium ( k ) current in the generation of prolonged qt and potential action duration has been noticed . ito is the transient outward k+ current which is one of the main repolarizing currents in the mammalian myocardium and is generally believed to flow through kv1.4 , kv4.2 and kv4.3 channels in rats [ 49 , 50 ] . in heart hypertrophic cases ito is down - regulated [ 51 - 53 ] . low ito density , kv1.4 and kv4.3 down - regulation in the left ventricle and prolonged action was noticed in the group treated with nandrolone compared to the control group . homogenous distribution of kv4.3 channel in the rat s ventricular wall , higher kv4.2 in the epicardial and lower in the endocardial ventricular wall have been observed [ 49 , 50 ] . these differences may partially explain the up - regulation of kv4.2 and prolonged qtc interval and action potential . the expression of kchip2 is considerably decreased in heart failure and hypertrophy [ 54 , 55 ] . kchip2 was found to be significant for ito expression in the human heart and the correlation between kchip2 absence and a total loss of ito together with an increased susceptibility to ventricular arrhythmias in mice has been shown . according to riezzo the following effects were produced in physically trained mice intramuscularly treated with nd : moderate increase of heart weight , morphologically extensive cardiac hypertrophy and a wide colliquative myocytolysis which together could result in a significant heart failure . medei et al . found approximately 25% less nuclei and higher cardiomyocyte nuclei diameter in the ventricles of the group treated with nd . lower nuclei suggests a toxic effect of nd which may involve a pro - apoptotic mechanism . tanno et al found that nd treatment whether combined with resistance training or not induced pathological concentric hypertrophy , re - expression of fetal genes , systolic and diastolic function impairment and an incremented myocardial collagen content leading to lvh . increased relative left ventricle wall thickness ( rwt ) was observed as a consequence of intensive physical training in rats treated with nd compared to the respective non - trained ones . in addition , the non - trained nandrolone treated group also produced higher rwt compared to the non - trained treated group . increased interventricular septum thickness in the end - diastole ( ivsdia ) was noticed in both the non - trained nandrolone treated and trained vehicle - treated groups , compared to the non - trained vehicle treated rats . a considerable lower ratio of maximum early to late transmitral flow velocity ( e / a ratio ) was observed in the trained groups , in comparison with non - trained groups . nandrolone - treated groups ( both trained and non - trained ) showed lower e / a ratio in comparison with the respective vehicle - treated groups . moreover , significant decrease in the expression of alpha - myosin heavy chain ( -mhc ) mrna and beta - myosin heavy chain ( -mhc ) mrna in the left ventricle was induced by nandrolone and resistance training respectively . . found that short - term nd treatment induces an overexpression of 2-adrenoceptors without cardiac hypertrophy . increment in cardiovascular mortality has been associated to an imbalance of ( ans ) activity . aass can acutely inhibit the reuptake of catecholamines into extraneuronal tissue and consequently the increment of catecholamine concentrations at receptor sites occurs . although , the neuronal catecholamine transporter is normally responsible for the reuptake of noradrenaline , it has also been proved responsible for nonexocytotic release of noradrenaline from sympathetic nerve terminals during ischemia . an increased release of noradrenaline has been implicated in ischemia - induced arrhythmia [ 63 , 64 ] . an increased activity of 6-phosphogluconate dehydrogenases and glucose-6-phosphate was observed in rat hearts , also nd activated isocitrate dehydrogenase and malic enzyme , which are other nadp - linked dehydrogenases . during the same study a significantly increased heart weight it was shown that treatment with nd causes small qrs complex extension that might slightly reduce the spreading rate of the action potential through the heart ventricles , possibly because of the greater heart mass . it is known that administration of doses higher than normal ( supraphysiological ) of nd impair exercise - induced cardioprotection in treadmill - exercised rats . chaves et al . were the first to say that enhanced levels of antioxidant enzyme levels produced after exercise are impaired with nd treatment ( 10mg / kg for 8 weeks ) , a fact which is well correlated to the cardiac injurious effects of the drug . it was observed that the hearts of animals treated with nandrolone and having undergone training ( dt group ) showed lower glutathione peroxidase ( gpx ) , superoxide dismutase ( sod ) and glutathione reductase ( gr ) activities compared with controls and trained groups of animals ( ct ) . the latter observation indicates that nandrolone could act through blocking or down regulating the mechanisms implicated in the improvement of antioxidant defenses in dt animals , which might explain the lower percentage of left ventricular developed pressure and augmented infarct size in dt group . it has been shown , in other researches on rats that exercise training improves myocardial resistance to reperfusion injury / ischemia [ 67 - 69 ] since physiological cardiac hypertrophy amends the sensitiveness of that heart making it more resistant to the previously mentioned disorders in vivo rat hearts . notwithstanding the strenuous research efforts , the molecular mechanism(s ) involved in exercise - induced cardio protection is still debatable . the numerous studies above reported in animal models , especially in rats , have called into question several pathophysiological mechanisms , which may explain some of the macroscopic and microscopic finding regarding the 19 cases here reported ; however , we have to underline that these cases are single case report or small series of cases and not experimental studies . moreover , the users of these substances frequently associate numerous steroids , in different forms , singularly and in several temporal combinations and cycles , and commonly , various steroid - accessory drugs are also used . therefore , the interpretation of the postmortem findings is particularly difficult and no comprehensive conclusions can be done . finally , a brief remark must be placed regarding the bmi which was in all cases ( 12 males and 2 females ) higher than 25 . although according to the bmi ranges from an overweight ( 9 cases ) to a very severely obese ( 2 cases ) were found , however , the bmi is not a direct measure of body fatness and bmi is calculated from an individual 's weight which includes both muscle and fat . as a result , some subjects such as highly trained athletes may have a high bmi because of increased muscularity rather than increased body fatness ; therefore , these results should be evaluated with caution . the relationship between aas abuse , vigorous exercise training , and cardiac death can be evaluated only by the application of an investigative protocol , which must include a rigorous methodology covering : a complete autopsy with a special regard to aas target organs and apparatus ( the cardiovascular system in primis ) . a broad toxicological investigation , preceded by a careful evaluation of clinico - anamnestic data , in order to confirm an aas consumption ( including type of aass , concentration and interval of exposure ) and the possible detection of other substances which could have contributed to the fatal outcome . for this purpose , different matrices can be used ; urine is the most common , because it provides a prolonged detection time window , but also several other matrices such as : blood , serum , plasma , hair , oral fluid and nails can be used . the comparison of the cases reported here , allows us to support the hypothesis that the combined effects of strong workout , the prolonged / chronic or previous abuse of aass in different forms and combinations , have predisposed these subjects to develop different patterns of myocardial injuries and consequent sudden cardiac death [ 21 , 23 ] . therefore , the authors would strengthen the warning already expressed in previous reports [ 21 , 23 ] against the use / abuse of these substances among professional and non - professional athletes . moreover , only through a careful examination of all suspicious cases of aas related deaths with the application of a rigorous investigative protocol , these cases can be identified and they could provide further information and data that may increase the knowledge of this type of deaths .

hypermethylation of the promoter region of the p16ink4a ( p16 ) gene plays a significant role in the development and progression of colorectal cancer ( crc ) . the aim of the present study was to establish the role of the methylation status of the p16 gene in 114 crc cases and to correlate it with the various clinicopathological parameters . analysis of p16 promoter methylation was performed by methylation - specific pcr . forty - eight ( 42.1% ) of the crc cases were found to be methylated for the p16 gene in our population . the methylation status was found to be associated with the gender , lymph node status , tumour stage , smoking status and tumour grade of the crc patients . p16 plays a pivotal role in tumour development and progression to advanced stages . anabolic androgenic steroids ( aass ) represent a large group of synthetic derivatives of testosterone , produced to maximize anabolic effects and minimize the androgenic ones . aas can be administered orally , parenterally by intramuscular injection and transdermally . androgens act by binding to the nuclear androgen receptor ( ar ) in the cytoplasm and then translocate into the nucleus . this binding results in sequential conformational changes of the receptor affecting the interaction between receptor and protein , and receptor and dna.skeletal muscle can be considered as the main target tissue for the anabolic effects of aas , which are mediated by ars which after exposure to aass are up - regulated and their number increases with body building . therefore , aass determine an increase in muscle size as a consequence of a dose - dependent hypertrophy resulting in an increase of the cross - sectional areas of both type i and type ii muscle fibers and myonuclear domains . moreover , it has been reported that aass can increase tolerance to exercise by making the muscles more capable to overload therefore shielding them from muscle fiber damage and improving the level of protein synthesis during recovery.despite some therapeutic use of aass , there is also wide abuse among athletes especially bodybuilders in order to improve their performances and to increase muscle growth and lean body mass , taking into account the significant anabolic effects of these drugs.the prolonged misuse and abuse of aass can determine several adverse effects , some of which may be even fatal especially on the cardiovascular system because they may increase the risk of sudden cardiac death ( scd ) , myocardial infarction , altered serum lipoproteins , and cardiac hypertrophy . the aim of this review is to focus on deaths related to aas abuse , trying to evaluate the autoptic , histopathological and toxicological findings in order to investigate the pathophysiological mechanism that underlines this type of death , which is still obscure in several aspects . the review of the literature allowed us to identify 19 fatal cases between 1990 and 2012 , in which the autopsy excluded in all cases , extracardiac causes of death .

in general relativity , kinetic theory has been used relatively sparsely to model phenomenological matter in comparison to fluid models , although interest has increased in recent years . from a mathematical point of view there are fundamental advantages to using a kinetic description . in non - curved spacetimes kinetic theory has been studied intensively as a mathematical subject during several decades , and it has also played an important role from an engineering point of view . the main purpose of this review paper is to discuss mathematical results for the einstein - vlasov system . however , in the first part of this introduction , we review kinetic theory in non - curved spacetimes and focus on the special - relativistic case , although some results in the non - relativistic case will also be mentioned . the reason that we focus on the relativistic case is not only that it is more closely related to the main theme in this review , but also that the literature on relativistic kinetic theory is very sparse in comparison to the non - relativistic case , in particular concerning the relativistic and non - relativistic boltzmann equation . we believe that a good understanding of kinetic theory in non - curved spacetimes is fundamental to good comprehension of kinetic theory in general relativity . moreover , it is often the case that mathematical methods used to treat the einstein - vlasov system are carried over from methods developed in the special relativistic or non - relativistic case . the purpose of kinetic theory is to model the time evolution of a collection of particles . for instance , the particles are atoms and molecules in a neutral gas or electrons and ions in a plasma . in astrophysics the particles are stars , galaxies or even clusters of galaxies . a characteristic feature of kinetic theory is that its models are statistical and the particle systems are described by density functions f = f(t , x , p ) , which represent the density of particles with given spacetime position ( t , x ) and momentum p. a density function contains a wealth of information , and macroscopic quantities are easily calculated from this function . in a fluid model the quantities that describe the system do not depend on the momentum p but only on the spacetime point ( t , x ) . a choice of model is usually made with regard to the physical properties of interest for the system or with regard to numerical considerations . it should be mentioned that a too naive fluid model may give rise to shell - crossing singularities , which are unphysical . in a kinetic description the time evolution of the system is determined by the interactions between the particles , which depend on the physical situation . for instance , the driving mechanism for the time evolution of a neutral gas is the collision between particles ( the boltzmann equation ) . for a plasma the interaction is through the electromagnetic field produced by the charges ( the vlasov - maxwell system ) , and in astrophysics the interaction is gravitational ( the vlasov - poisson system and the einstein - vlasov system ) . of course , combinations of interaction processes are also considered but in many situations one of them is strongly dominating and the weaker processes are neglected . consider a collection of neutral particles in minkowski spacetime . let the signature of the metric be ( , + , + , + ) . in this section we assume that all the particles have rest mass m = 1 , and we normalize the speed of light c to one . we point out that in section 2 on the einstein - vlasov system , the dependence on the rest mass and the speed of light will be included in the formulation of the system . the four - momentum of a particle is denoted by p , a = 0 , 1 , 2 , 3 . since all particles have equal rest mass , the four - momentum for each particle is restricted to the mass shell , ppa = m = 1 . thus , by denoting the three - momentum by p , p may be written p = ( p , p ) , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${p^0 } = \sqrt { 1 + \vert p{\vert ^2}}$\end{document } is the energy of a particle with three - momentum p , and p is the usual euclidean length of p. the relativistic velocity of a particle with momentum p is denoted by p and is given by 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat p = { p \over { \sqrt { 1 + \vert p\vert ^{2}}}}.$$\end{document } note that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\vert \hat p\vert < 1 = c$\end{document}. the relativistic boltzmann equation models the spacetime behavior of the one - particle distribution function f = f(t , x , p ) , and it has the form 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left({{\partial _ t } + { p \over { { p^0 } } } \cdot { \nabla _ x } } \right)f = q(f , f),$$\end{document } where the relativistic collision operator q(f , g ) is defined by 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(f , g ) = \int\nolimits_{{{\mathbb r}^2 } } { \int\nolimits_{{{\mathbb s}^2 } } { k(p , q,\omega)[f(p + a(p , q,\omega)\omega)g(q - a(p , q,\omega)\omega ) - f(p)g(q)]d\omega \;dp.}}$$\end{document } note that g = f in equation ( 2 ) . here d is the element of surface area on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\rm{{\mathbb s}}}^2}$\end{document } and k(p , q , ) is the scattering kernel , which depends on the differential cross - section in the interaction process . we refer to , and for examples of differential cross - sections in the relativistic case . if two particles , with momentum p and q respectively , collide elastically with scattering angle \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\omega \in { { \rm{{\mathbb s}}}^2}$\end{document } , their momenta will change , i.e. , p p and q q. the relation between p , q and p , q is given by 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p^{\prime } = p + a(p , q,\omega)\omega , \quad q^{\prime } = q - a(p , q,\omega)\omega,$$\end{document } where 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a(p , q,\omega ) = { { 2({p^0 } + { q^0}){p^0}{q^0}(\omega \cdot ( \hat q - \hat p ) ) } \over { { { ( { p^0 } + { q^0})}^2 } - { { ( \omega \cdot ( p + q))}^2}}}.$$\end{document } this relation is a consequence of four - momentum conservation , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^a } + { q^a } = { p^{a^{\prime } } } + { q^{a{\prime}}},$$\end{document } or equivalently 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^0 } + { q^0 } = { p^{0{\prime } } } + { q^{0{\prime}}},$$\end{document } 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p + q = { p^{\prime } } + { q^{\prime}}.$$\end{document } these are the conservation equations for relativistic particle dynamics . in the classical case the corresponding conservation equations read 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert p\vert ^{2 } + \vert q\vert ^{2 } = \vert p^{\prime}\vert ^{2 } + \vert q^{\prime}\vert ^{2},$$\end{document } 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p + q = { p^{\prime } } + { q^{\prime}}.$$\end{document } the function ( p , q , ) gives the distance between p and p ( q and q ) in momentum space , and the analogue function in the non - relativistic , newtonian , classical case has the form 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_{{\rm{cl}}}}(p , q,\omega ) = \omega \cdot ( q - p).$$\end{document } by inserting ac1 in place of a in equation ( 3 ) we obtain the classical boltzmann collision operator ( disregarding the scattering kernel , which is also different ) . we point out that there are other representations of the collision operator ( 3 ) , cf . . in and classical solutions to the relativistic boltzmann equations are studied as c , and it is proven that the limit as c of these solutions satisfies the classical boltzmann equation . the former work is more general since general initial data is considered , whereas the latter is concerned with data near vacuum . the latter result is stronger in the sense that the limit , as c , is shown to be uniform in time . the main result concerning the existence of solutions to the classical boltzmann equation is a theorem by diperna and lions that proves existence , but not uniqueness , of renormalized solutions . an analogous result holds in the relativistic case , as was shown by dudyski and ekiel - jeewska , cf . also . regarding classical solutions , illner and shinbrot have shown global existence of solutions to the non - relativistic boltzmann equation for initial data close to vacuum . glassey showed global existence for data near vacuum in the relativistic case in a technical work . he only requires decay and integrability conditions on the differential cross - section , although these are not fully satisfactory from a physics point of view . by imposing more restrictive cut - off assumptions on the differential cross - section , strain gives a different proof , which is more related to the proof in the non - relativistic case than is . for the homogeneous relativistic boltzmann equation , global existence for small initial data has been shown in under the assumption of a bounded differential cross - section . for initial data close to equilibrium , global existence of classical solutions has been proven by glassey and strauss using assumptions on the differential cross - section , which fall into the regime hard potentials , whereas strain has shown existence in the case of soft potentials . in the non - relativistic case , we refer to [ 189 , 172 , 119 ] for analogous results . the collision operator q(f , g ) may be written in an obvious way as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(f , g ) = { q^ + } ( f , g ) - { q^ -}(f , g),$$\end{document } where q and q are called the gain and loss term , respectively . if the loss term is deleted the gain - term - only boltzmann equation is obtained . it is interesting to note that the methods of proof for the small data results mentioned above concentrate on gain - term - only equations , and once that is solved it is easy to include the loss term . in it is shown that the gain - term - only classical and relativistic boltzmann equations blow up for initial data not restricted to a small neighborhood of trivial data . thus , if a global existence proof of classical solutions for unrestricted data will be given , it will necessarily use the full collision operator . the gain term has a nice regularizing property in the momentum variable . in it is proven that given f l( ) and g l( ) with f , g 0 , then 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\vert \vert { q^ + } ( f , g)\vert \vert _ { { { h^1}({\mathbb r}_p^3 ) } } \leq c\vert \vert f\vert \vert _ { { { l^2}({\mathbb r}_p^3)}}\vert \vert g\vert \vert _ { { { l^1}({\mathbb r}_p^3)}},$$\end{document } under some technical requirements on the scattering kernel . here h is the usual sobolev space . the proof relies on the theory of fourier integral operators and on the method of stationary phase , and requires a careful analysis of the collision geometry , which is very different in the relativistic case . simplified proofs in the classical and relativistic case are given in [ 193 , 194 ] . more precisely , lions used the regularizing theorem to prove that solutions to the classical boltzmann equation , with periodic boundary conditions , converge in l to a global maxwellian , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m = { e^{- \alpha \vert p\vert ^{2 } + \beta \cdot p + \gamma}}\quad { \rm{with}}\;\alpha , \gamma \in r,\quad \alpha > 0,\quad \beta \in { { \mathbb r}^3},$$\end{document } as time goes to infinity . it should be pointed out that the convergence takes place through a sequence of times tending to infinity and it is not known whether the limit is unique or depends on the sequence . in the relativistic situation , the analogous question of convergence to a relativistic maxwellian , or a jttner equilibrium solution , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j = { e^{- \alpha \sqrt { 1 + \vert p\vert ^{2 } } + \beta \cdot p + \gamma}},\quad \alpha , \beta , \;{\rm{and}}\;\gamma \;{\rm{as}}\;{\rm{above}},\;{\rm{with}}\;\alpha > \vert \beta \vert,$$\end{document } was studied by glassey and strauss [ 87 , 88 ] . in the periodic case , they proved convergence in a variety of function spaces for initial data close to a jttner solution . having obtained the regularizing theorem for the relativistic gain term , it is a straightforward task to follow the method of lions and prove convergence to a global jttner solution for arbitrary initial data ( satisfying the natural bounds of finite energy and entropy ) , which are periodic in the space variables , cf . . we also mention that in the non - relativistic case desvillettes and villani have studied the convergence rate to equilibrium in detail . a similar study in the relativistic case has not yet been achieved . for more information on the relativistic boltzmann equation on minkowski space we refer to [ 54 , 68 , 181 , 79 ] and in the non - relativistic case we refer to [ 190 , 79 , 53 ] . let us consider a collision - less plasma , which is a collection of particles for which collisions are relatively rare and the interaction is through their charges . for simplicity we assume that the plasma consists of one type of particle , although the results below hold for plasmas with several particle species . the particle rest mass and the particle charge are normalized to one . in the kinetic framework , the most general set of equations for modeling a collision - less plasma is the relativistic vlasov - maxwell system : 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + \hat v \cdot { \nabla _ x}f + ( e(t , x ) + \hat v \times b(t , x ) ) \cdot { \nabla _ v}f = 0$$\end{document } 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ \quad \nabla \cdot e = \rho,$$\end{document } 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}b = - c\nabla \times e,\quad \quad \nabla \cdot b = 0.$$\end{document } the notation follows the one already introduced with the exception that the momenta are now denoted by v instead of p. this has become a standard notation in this field . e and b are the electric and magnetic fields , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat \upsilon}$\end{document } is the relativistic velocity , 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat v = { v \over { \sqrt { 1 + \vert v\vert ^{2}/{c^2}}}},$$\end{document } where is the speed of light . the charge density and current j are given by 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho = \int\nolimits_{{{\mathbb r}^3 } } { fdv,\quad j = } \int\nolimits_{{{\mathbb r}^3 } } { \hat vfdv.}$$\end{document } equation ( 12 ) is the relativistic vlasov equation and equations ( 13 , 14 ) are the maxwell equations . a special case in three dimensions is obtained by considering spherically - symmetric initial data . for such data it can be shown that the solution will also be spherically symmetric , and that the magnetic field has to be constant . the maxwell equation e = tb then implies that the electric field is the gradient of a potential . hence , in the spherically - symmetric case the relativistic vlasov - maxwell system takes the form 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + \hat v \cdot { \nabla _ x}f + \beta e(t , x ) \cdot { \nabla _ v}f = 0,$$\end{document } 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = \nabla \phi , \quad \nabla \phi = \rho.$$\end{document } here = 1 , and the constant magnetic field has been set to zero , since a constant field has no significance in this discussion . this system makes sense for any initial data , without symmetry constraints , and is called the relativistic vlasov - poisson system . another special case of interest is the classical limit , obtained by letting c in equations ( 12 , 13 , 14 ) , yielding : 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + v \cdot { \nabla _ x}f + \beta e(t , x ) \cdot { \nabla _ v}f = 0,$$\end{document } 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = \nabla \phi , \quad \nabla \phi = \rho,$$\end{document } where = 1 . this is the vlasov - poisson system , and = 1 corresponds to repulsive forces ( the plasma case ) . taking = 1 means attractive forces and the vlasov - poisson system is then a model for a newtonian self - gravitating system . one of the fundamental problems in kinetic theory is to find out whether or not spontaneous shock formations will develop in a collision - less gas , i.e. , whether solutions to any of the equations above will remain smooth for all time , given smooth initial data . if the initial data are small this problem has an affirmative solution in all cases considered above [ 81 , 86 , 32 , 33 ] . for initial data unrestricted in size the picture is more involved . in order to obtain smooth solutions globally in time , the main issue is to control the support of the momenta 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) : = \sup \{\vert v\vert:\exists ( s , x ) \in [ 0,t ] \times { { \mathbb r}^3}\;{\rm{such}}\;{\rm{that}}\;f(s , x , v ) \neq 0\},$$\end{document } i.e. , to bound q(t ) by a continuous function so that q(t ) will not blow up in finite time . that such a control is sufficient for obtaining global existence of smooth solutions follows from well - known results in the different cases , cf . [ 85 , 104 , 39 , 96 , 34 , 81 ] . for the full three - dimensional relativistic vlasov - maxwell system , the problem of establishing whether or not solutions will remain smooth for all time is open . a different sufficient criterion for global existence in this case is given by pallard in , and he also shows a new bound for the electromagnetic field in terms of q(t ) in . in two space and three momentum dimensions , glassey and schaeffer [ 82 , 83 ] have shown that q(t ) can be controlled for the relativistic vlasov - maxwell system , which thus yields global existence of smooth solutions in that case . the relativistic and non - relativistic vlasov - poisson equations are very similar in form . however , the mathematical results concerning the two systems are very different . in the non - relativistic case , batt gave an affirmative solution in 1977 in the case of spherically - symmetric data . pfaffelmoser was the first one to give a proof for general smooth data . a simplified version of the proof is given by schaeffer in . pfaffelmoser obtained the bound \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq c{(1 + t)^{(51 + \delta)/11}},$$\end{document } where > 0 can be taken as arbitrarily small . the sharpest bound valid for = 1 and = 1 has been given by horst and reads \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq c(1 + t)\log ( 2 + t).$$\end{document } in the case of repulsive forces ( = 1 ) rein has found a better estimate by using a new identity for the vlasov - poisson system , discovered independently by illner and rein and by perthame . rein s estimate reads \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq c{(1 + t)^{2/3}}.$$\end{document } independently , and at about the same time as pfaffelmoser gave his proof , lions and perthame used a different method for proving global existence . their method is more generally applicable , and the two studies and are examples of problems in related systems , where their method has been successful . on the other hand , their method does not give such strong growth estimates on q(t ) as described above . for the relativistic vlasov - poisson equation , glassey and schaeffer showed in the case = 1 that if the data are spherically symmetric , q(t ) can be controlled , which is analogous to the result by batt mentioned above . also in the case of cylindrical symmetry they are able to control q(t ) ; see . if = 1 it was shown in that blow - up occurs in finite time for spherically - symmetric data with negative total energy . more recently , lemou et al . have investigated the structure of the blow - up solution . they show that the blow - up is determined by the self - similar solution of the ultra - relativistic gravitational vlasov - poisson system . it should be pointed out that the relativistic vlasov - poisson system is unphysical since it lacks the lorentz invariance ; it is a hybrid of a classical galilei invariant field equation and a relativistic transport equation ( 17 ) , cf . . in particular , in the case = 1 , it is not a special case of the einstein - vlasov system . only for spherically - symmetric data , in the case the situation for weak solutions is different , in particular the existence of weak solutions to the relativistic vlasov - maxwell system is known [ 70 , 139 ] . we also mention that models , which take into account both collisions and the electric and magnetic fields generated by the particles have been investigated . classical solutions near a maxwellian for the vlasov - maxwell - boltzmann system are constructed by guo in . a similar result for the vlasov - maxwell - landau system near a jttner solution is shown by guo and strain in . we refer to the book by glassey and the review article by rein for more information on the relativistic vlasov - maxwell system and the vlasov - poisson system . before turning to the main theme of this review , i.e. , the einstein - vlasov system nordstrm gravity is an alternative theory of gravity introduced in 1913 . by coupling this model to a kinetic description of matter the nordstrm - vlasov system results . in nordstrm gravity the nordstrm - vlasov system reads 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\partial _ t^2\phi - { \delta _ x}\phi = - { e^{4\phi}}\int\nolimits_{{{\mathbb r}^3 } } { { { { \mathfrak f}dp } \over { \sqrt { 1 + \vert p\vert ^{2}}}},}$$\end{document } 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}{\mathfrak f } + \hat{p } \cdot { \nabla _ x}{\mathfrak f } - \left [ { ( { \partial _ t}\phi + \hat{p } \cdot { \nabla _ x}\phi)p + { { ( 1 + \vert p\vert ^{2})}^{- 1/2}}{\nabla _ x}\phi } \right ] \cdot { \nabla _ p}{\mathfrak f } = 0.$$\end{document } here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\hat{p } = { p \over { \sqrt { 1 + \vert p\vert ^{2}}}},$$\end{document } denotes the relativistic velocity of a particle with momentum p. the mass of each particle , the gravitational constant , and the speed of light are all normalized to one . a solution ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({\mathfrak f},\phi)$\end{document } , ) of this system is interpreted as follows . the spacetime is a lorentzian manifold with a conformally - flat metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g_{\mu \nu } = e^{2\phi}{\rm diag}(- 1,1,1,1).$$\end{document}. the particle distribution f defined on the mass shell in this metric is given by 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(t , x , p ) = { \mathfrak f}(t , x,{e^\phi}p).$$\end{document } the first mathematical study of this system was initiated by calogero in , where the existence of static solutions is established . although the nordstrom - vlasov model of gravity does not describe physics correctly , the system approaches the vlasov - poisson system in the classical limit . indeed , it is shown in that solutions of the nordstrm - vlasov system tend to solutions of the vlasov - poisson system as the speed of light goes to infinity . the cauchy problem was studied by several authors [ 51 , 50 , 15 , 108 , 131 ] and the question of global existence of classical solutions for general initial data was open for some time . another interesting result for the nordstrm - vlasov system is given in , where a radiation formula , similar to the dipole formula in electrodynamics , is rigorously derived . in this section we consider a self - gravitating collision - less gas in the framework of general relativity and we present the einstein - vlasov system . it is most often the case in the mathematics literature that the speed of light c and the gravitational constant g are normalized to one , but we keep these constants in the formulas in this section since in some problems they do play an important role . however , in most of the problems discussed in the forthcoming sections these constants will be normalized to one . let m be a four - dimensional manifold and let gab be a metric with lorentz signature ( , + , + , + ) so that ( m , gab ) is a spacetime . the metric is assumed to be time - orientable so that there is a distinction between future and past directed vectors . the possible values of the four - momentum p of a particle with rest mass m belong to the mass shell pm tm , defined by 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_m } : = \{({x^a},{p^a } ) \in tm:{g_{ab}}({x^a}){p^a}{p^b } = - { m^2}{c^2},{p^a}\;{\rm{is}}\;{\rm{future}}\;{\rm{directed}}\}.$$\end{document } hence , if m > 0 , pm(x ) is the set of all future - directed time - like vectors with length cm , and if m = 0 it is the set of all future - directed null vectors . on pm we take ( x , p ) , a = 0 , 1 , 2 , 3 and j = 1 , 2 , 3 ( letters in the beginning of the alphabet always take values 0 , 1 , 2 , 3 and letters in the middle take 1 , 2 , 3 ) as local coordinates , and p is expressed in terms of p and the metric in view of equation ( 25 ) . below we drop the index m on fm and simply write f. since we are considering a collisionless gas , the particles follow the geodesics in spacetime . the geodesics are projections onto spacetime of the curves in pm defined in local coordinates by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { d{x^a } } \over { ds } } = { p^a}},\\ { { { d{p^j } } \over { ds } } = - \gamma _ { bc}^j{p^b}{p^c}}.\\ \end{array}$$\end{document } here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma _ { bc}^a$\end{document } are the christoffel symbols . along a geodesic the density function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f = f({x^a},{p^j})$\end{document } is invariant so that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${d \over { ds}}f({x^a}(s),{p^j}(s ) ) = 0,$$\end{document } which implies that 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^a}{{\partial f } \over { \partial { x^a } } } - \gamma _ { ab}^j{p^a}{p^b}{{\partial f } \over { \partial { p^j } } } = 0.$$\end{document } this is accordingly the vlasov equation . we point out that sometimes the density function is considered as a function on the entire tangent bundle tm rather than on the mass shell pm tm . the vlasov equation for f = f(x , p ) then takes the form 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p^a}{{\partial f } \over { \partial { x^a } } } - \gamma _ { bc}^a{p^b}{p^c}{{\partial f } \over { \partial { p^a } } } = 0.$$\end{document } this equation follows from ( 26 ) if we take the mass shell condition gabpp = mc into account . indeed , by abuse of notation , we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \partial f } \over { \partial { x^a } } } = { { \partial f } \over { \partial { x^a } } } + { { \partial f } \over { \partial { p^0}}}{{\partial { p^0 } } \over { \partial { x^a}}},}\\ { { { \partial f } \over { \partial { p^j } } } = { { \partial f } \over { \partial { p^j } } } + { { \partial f } \over { \partial { p^0}}}{{\partial { p^0 } } \over { \partial { p^j}}}.}\\ \end{array}$$\end{document } here f is considered as a function on pm in the left - hand side , and on tm in the right - hand side . from the mass shell condition gabpp = mc we derive \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \partial { p^0 } } \over { \partial { x^a } } } = - { 1 \over { { p_0}}}{p^b}{p_c}\gamma _ { ab}^c,}\\ { { { \partial { p^0 } } \over { \partial { p^j } } } = - { { { p_j } } \over { { p_0}}}.}\\ \end{array}$$\end{document } inserting these relations into ( 26 ) we obtain ( 27 ) . if we let t = x , and divide the vlasov equation ( 26 ) by p we obtain the most common form in the literature of the vlasov equation 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\partial f } \over { \partial t } } + { { { p^j } } \over { { p^0}}}{{\partial f } \over { \partial { x^j } } } - { 1 \over { { p^0}}}\gamma _ { ab}^j{p^a}{p^b}{{\partial f } \over { \partial { p^j } } } = 0.$$\end{document } in a fixed spacetime the vlasov equation ( 28 ) is a linear hyperbolic equation for f and we can solve it by solving the characteristic system , 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{x^i } } \over { ds } } = { { { p^i } } \over { { p^0}}},$$\end{document } 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{d{p^i } } \over { ds } } = - \gamma _ { ab}^i{{{p^a}{p^b } } \over { { p^0}}}.$$\end{document } in terms of initial data f0 the solution of the vlasov equation can be written as 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f({x^a},{p^i } ) = { f_0}({x^i}(0,{x^a},{p^i}),{p^i}(0,{x^a},{p^i})),$$\end{document } where x(s , x , p ) and p(s , x , p ) solve equations ( 29 , 30 ) , and where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${x^i}(t,{x^a},{p^i } ) = { x^i}\;{\rm{and}}\;{p^i}(t,{x^a},{p^i } ) = { p^i}.$$\end{document } in order to write down the einstein - vlasov system we need to know the energy - momentum tensor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_{ab}^m$\end{document } in terms of f and gab . we define 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{ab}^m = c\sqrt { \vert { g_{ab}}\vert } \int\nolimits_{{{\mathbb r}^3 } } { f{p_a}{p_b}{{d{p^1}d{p^2}d{p^3 } } \over { - { p_0}}}},$$\end{document } where , as usual , pa = gabp , and gab denotes the absolute value of the determinant of gab . we remark that the measure \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mu : = { { \sqrt { \vert { g_{ab}}\vert } } \over { - { p_0}}}d{p^1}d{p^2}d{p^3},$$\end{document } is the induced metric of the submanifold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${p_m}({x^a } ) \subset { t_{{x^a}}}m$\end{document } , and that is invariant under lorentz transformations of the tangent space , and it is often the case in the literature that is written as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t_{ab}^m = c\int\nolimits_{{p_m}({x^a } ) } { f{p_a}{p_b}\mu.}$$\end{document } let us now consider a collisionless gas consisting of particles with different rest masses m1 , m2 , , mn , described by n density functions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f_{{m_j}}}$\end{document } , j = 1 , , n. then the vlasov equations for the different density functions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f_{{m_j}}}$\end{document } , together with the einstein equations , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{ab } } - { 1 \over 2}r{g_{ab } } + \lambda { g_{ab } } = { { 8\pi g } \over { { c^4}}}\sum\limits_{k = 1}^n { t_{ab}^{{m_k}},}$$\end{document } form the einstein - vlasov system for the collision - less gas . here rab is the ricci tensor , r is the scalar curvature and is the cosmological constant . henceforth , we always assume that there is only one species of particles in the gas and we write tab for its energy momentum tensor . moreover , in what follows , we normalize the rest mass m of the particles , the speed of light c , and the gravitational constant g , to one , if not otherwise explicitly stated that this is not the case . we define the particle current density \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n^a } = - \int\nolimits_{{{\mathbb r}^3 } } { f{p^a } } \sqrt { \vert { g_{ab}}\vert } { { d{p^1}d{p^2}d{p^3 } } \over { { p_0}}}.$$\end{document } using normal coordinates based at a given point and assuming that f is compactly supported , it is not hard to see that tab is divergence - free , which is a necessary compatibility condition since the left - hand side of ( 2 ) is divergence - free by the bianchi identities . a computation in normal coordinates also shows that n is divergence - free , which expresses the fact that the number of particles is conserved . if v is a future - directed time - like or null vector then we have nav 0 with equality if and only if f = 0 at the given point . hence , n is always future - directed time - like , if there are particles at that point . moreover , if v and w are future - directed time - like vectors then tabvw 0 , which is the dominant energy condition . this is called the non - negative pressure condition , and it implies that the strong energy condition holds as well . that the energy conditions hold for vlasov matter is one reason that the vlasov equation defines a well - behaved matter model in general relativity . another reason is the well - posedness theorem by choquet - bruhat for the einstein - vlasov system that we state below . before stating that theorem we first discuss the conditions imposed on the initial data . the initial data in the cauchy problem for the einstein - vlasov system consist of a 3-dimensional manifold s , a riemannian metric gij on s , a symmetric tensor kij on s , and a non - negative scalar function f0 on the tangent bundle ts of s. the relationship between a given initial data set ( gij , kij ) on s and the metric gab on the spacetime manifold is , that there exists an embedding of s into the spacetime such that the induced metric and second fundamental form of (s ) coincide with the result of transporting ( gij , kij ) with . for the relation of the distribution functions f and f0 we have to note that f is defined on the mass shell . the initial condition imposed is that the restriction of f to the part of the mass shell over (s ) should be equal to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${f_0 } \circ ( { \psi ^{- 1}},d{(\psi)^{- 1 } } ) \circ \phi$\end{document } , where sends each point of the mass shell over (s ) to its orthogonal projection onto the tangent space to (s ) . an initial data set for the einstein - vlasov system must satisfy the constraint equations , which read 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r - { k_{ij}}{k^{ij } } + { ( { \rm{tr}}\;k)^2 } = 16\pi \rho,$$\end{document } 34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla _ i}k_l^i - { \nabla _ l}({\rm{tr}}\;k ) = 8\pi j_{l}.$$\end{document } here = tabnn j = htbcn , where n is the future directed unit normal vector to the initial hypersurface , and h = g + nn is the orthogonal projection onto the tangent space to the initial hypersurface . in terms of f0 we can express and j by ( j satisfies naj = 0 , so it can naturally be identified with a vector intrinsic to s ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \rho = - \int\nolimits_{{{\mathbb r}^3 } } { { f_0}{p^a}{p_a } } \sqrt { \vert { g_{ij}}\vert } { { d{p^1}d{p^2}d{p^3 } } \over { 1 + { p_j}{p^j}}},}\\ { { j_l } = \int\nolimits_{{{\mathbb r}^3 } } { { f_0}{p_l } } \sqrt { \vert { g_{ij}}\vert } d{p^1}d{p^2}d{p^3}.}\\ \end{array}$$\end{document } we can now state the local existence theorem by choquet - bruhat , for the einstein - vlasov system . theorem 1 let s be a 3-dimensional manifold , gij a smooth riemannian metric on s , kij a smooth symmetric tensor on s and f0 a smooth non - negative function of compact support on the tangent bundle ts of s. suppose that these objects satisfy the constraint equations ( 33 , 34 ) . then there exists a smooth spacetime ( m , gab ) , a smooth distribution function f on the mass shell of this spacetime , and a smooth embedding into m , which induces the given initial data on s such that gab and f satisfy the einstein - vlasov system and (s ) is a cauchy surface . moreover , given any other spacetime ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({m^\prime},g_{ab}^\prime)$\end{document } ) , distribution function f and embedding satisfying these conditions , there exists a diffeomorphism from an open neighborhood of if (s ) in m to an open neighborhood of (s ) in m , which satisfies and carries gab and f to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$g_{ab}^\prime$\end{document } and f , respectively . the above formulation is in the case of smooth initial data ; for information on the regularity needed on the initial data we refer to and . in this context we also mention that local existence has been proven for the yang - mills - vlasov system in , and that this problem for the einstein - maxwell - boltzmann system is treated in . however , this result is not complete , as the non - negativity of f is left unanswered . also , the hypotheses on the scattering kernel in this work leave some room for further investigation . the local existence problem for physically reasonable assumptions on the scattering kernel does not seem well understood in the context of the einstein - boltzmann system , and a careful study of this problem would be desirable . the mathematical study of the einstein - boltzmann system has been very sparse in the last few decades , although there has been some activity in recent years . since most questions on the global properties are completely open let us only very briefly mention some of these works . global existence for the homogeneous einstein - boltzmann system in robertson - walker spacetimes is proven in , and a generalization to bianchi type i symmetry is established in . in the following sections we present results on the global properties of solutions of the einstein - vlasov system , which have been obtained during the last two decades . before ending this section we mention a few other sources for more background on the einstein - vlasov system , cf . in this section , we discuss results on global existence and on the asymptotic structure of solutions of the cauchy problem in the asymptotically - flat case . in general relativity two classes of initial data are distinguished in the study of the cauchy problem : asymptotically - flat initial data and cosmological initial data . the initial hypersurface is topologically and appropriate fall - off conditions are imposed to ensure that far away from the body spacetime is approximately flat . spacetimes , which possess a compact cauchy hypersurface , are called cosmological spacetimes , and data are accordingly given on a compact 3-manifold . in this case , the whole universe is modeled rather than an isolated body . one can also consider a case , which is un - physical , in which spacetime is asymptotically flat except in one direction , namely cylindrically - symmetric spacetimes , cf . the majority of the work so far has been devoted to the spherically - symmetric case but recently a result on static axisymmetric solutions has been obtained . in contrast to the asymptotically - flat case , cosmological spacetimes admit a large number of symmetry classes . this provides the possibility to study many special cases for which the difficulties of the full einstein equations are reduced . the main goal of these studies is to provide an answer to the weak and strong cosmic censorship conjectures , cf . the study of the global properties of solutions to the spherically - symmetric einstein - vlasov system was initiated two decades ago by rein and rendall , cf . also [ 135 , 156 ] . they chose to work in coordinates where the metric takes the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - { e^{2\mu ( t , r)}}d{t^2 } + { e^{2\lambda ( t , r)}}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\varphi ^2}),$$\end{document } where t , r 0 , [ 0, ] , [ 0 , 2 ] . these are called schwarzschild coordinates . asymptotic flatness is expressed by the boundary conditions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset{r \rightarrow \infty}{\lim } \lambda ( t , r ) = \underset{r \rightarrow \infty}{\lim } \mu ( t , r ) = 0,\quad \forall t \geq 0.$$\end{document } a regular center is also required and is guaranteed by the boundary condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda ( t,0 ) = 0\quad \forall t \geq 0.$$\end{document } the coordinates ( r , , ) give rise to difficulties at r = 0 and it is advantageous to use cartesian coordinates . with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x = ( r\sin \phi \cos \theta , r\sin \phi \sin \theta , r\cos \phi)$$\end{document } as spatial coordinates and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${v^j } = { p^j } + ( { e^\lambda } - 1){{x \cdot p } \over r}{{{x^j } } \over r}$$\end{document } as momentum coordinates , the einstein - vlasov system reads 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}f + { e^{\mu - \lambda}}{v \over { \sqrt { 1 + \vert v\vert ^{2 } } } } \cdot { \nabla _ x}f - \left({{\lambda _ t}{{x \cdot v } \over r } + { e^{\mu - \lambda}}{\mu _ r}\sqrt { 1 + \vert v\vert ^{2 } } } \right){x \over r } \cdot { \nabla _ v}f = 0,$$\end{document } 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\lambda _ r } - 1 ) + 1 = 8\pi { r^2}\rho,$$\end{document } 37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\mu _ r } + 1 ) - 1 = 8\pi { r^2}p,$$\end{document } 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\lambda _ t } = - 4\pi r{e^{\lambda + \mu}}j,$$\end{document } 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}({\mu _ { rr } } + ( { \mu _ r } - { \lambda _ r})({\mu _ r } + { 1 \over r } ) ) - { e^{- 2\mu}}({\lambda _ { tt } } + { \lambda _ t}({\lambda _ t } - { \mu _ t } ) ) = 8\pi { p_t}.$$\end{document } the matter quantities are defined by 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho ( t , x ) = \int\nolimits_{{{\mathbb r}^3 } } { \sqrt { 1 + \vert v\vert ^{2 } } f(t , x , v)dv,}$$\end{document } 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(t , x ) = \int\nolimits_{{{\mathbb r}^3 } } { { { \left({{{x \cdot v } \over r } } \right)}^2}f(t , x , v){{dv } \over { \sqrt { 1 + \vert v\vert ^{2}}}},}$$\end{document } 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$j(t , x ) = \int\nolimits_{{{\mathbb r}^3 } } { { { x\cdot v } \over r}f(t , x , v)dv,}$$\end{document } 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${p_t}(t , x ) = { 1 \over 2}\int\nolimits_{{{\mathbb r}^3 } } { \vert { { x \times v } \over r}\vert ^{2}f(t , x , v)dv.}$$\end{document } here is the energy density , j the current , p the radial pressure , and pt the tangential pressure . let us point out that these equations are not independent , e.g. , equations ( 38 ) and ( 39 ) follow from ( 35)(37 ) . as initial data we take a spherically - symmetric , non - negative , and continuously diferentiable function f0 with compact support that satisfies 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{\vert y\vert < r } { \int\nolimits_{{{\mathbb r}^3 } } { \sqrt { 1 + \vert v{\vert ^2 } } { f_0}(y , v)dv\;dy\ ; < { r \over 2}.}}$$\end{document } this condition guarantees that no trapped surfaces are present initially . the schwarzschild coordinates have the advantage that the resulting system of equations can be written in a quite condensed form . moreover , for most initial data , solutions are expected to exist globally in schwarzschild time , which sometimes is called the polar time gauge . let us point out here that there are initial data leading to spacetime singularities , cf . hence , the question of global existence for general initial data is only relevant if the time slicing of the spacetime is expected to be singularity avoiding , which is the case for schwarzschild time . however , one disadvantage is that these coordinates only cover a relatively small part of the spacetime , in particular trapped surfaces are not admitted . hence , to analyze the black - hole region of a solution these coordinates are not appropriate . here we only mention the other coordinates and time gauges that have been considered in the study of the spherically symmetric einstein - vlasov system . the einstein - vlasov system is investigated in double null coordinates in [ 64 , 63 ] . maximal - areal coordinates and eddington - finkelstein coordinates are used in [ 21 , 17 ] , and in respectively . in it is shown that for initial data satisfying ( 44 ) there exists a unique , continuously - differentiable solution f with f(0 ) = f0 on some right maximal interval [ 0 , t ) . if the solution blows up in finite time , i.e. , if t < , then (t ) becomes unbounded as t t. moreover , a continuation criterion is shown that says that a local solution can be extended to a global one , provided q(t ) can be bounded on [ 0 , t ) , where 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t):\sup \{\vert v\vert : \exists ( s , x ) \in [ 0,t ] \times { { \mathbb r}^3}\;{\rm{such}}\;{\rm{that}}\;f(s , x , v ) \neq 0\}.$$\end{document } this is analogous to the situation for the vlasov - maxwell system . a control of the v - support immediately implies that and p are bounded in view of equations ( 40 , 41 ) . in the vlasov - maxwell case the field equations have a regularizing effect in the sense that derivatives can be expressed through spatial integrals , and it follows that the derivatives of f can also be bounded if the v - support is bounded . for the einstein - vlasov system such a regularization is less clear , since , e.g. , r depends on p in a point - wise manner . however , in view of equation ( 39 ) certain combinations of second and first order derivatives of the metric components can be expressed in terms of the matter component pt , which is a consequence of the geodesic deviation equation . this fact turns out to also be sufficient for obtaining bounds on the derivatives of f , cf . [ 142 , 135 , 156 ] for details . the local existence result discussed above holds for compactly - supported initial data . the compact support condition in the momentum variables is in replaced by the fall - off condition 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset{(x , v ) \in { { \mathbb r}^6}}{\sup}{(1 + \vert v\vert)^5}\vert \overset{\circ}{f}(x , v)\vert < \infty.$$\end{document } we also refer to where a subclass of non - compactly - supported data is treated . local existence of solutions in double null coordinates and in eddington - finkelstein coordinates is established in , and respectively . in the authors also consider the problem of global existence in schwarzschild coordinates for small initial data for massive particles . they show that for such data the v - support is bounded on [ 0 , t ) . hence , the continuation criterion implies that t = . the resulting spacetime in is geodesically complete , and the components of the energy - momentum tensor as well as the metric quantities decay with certain algebraic rates in t. the mathematical method used by rein and rendall is inspired by the analogous small data result for the vlasov - poisson equation by bardos and degond . this should not be too surprising since for small data the gravitational fields are expected to be small and a newtonian spacetime should be a fair approximation . in this context we point out that in it is proven that the vlasov - poisson system is indeed the non - relativistic limit of the spherically - symmetric einstein - vlasov system , i.e. , the limit when the speed of light c . in this result is shown without symmetry assumptions . as mentioned above the local and global existence problem has been studied using other time gauges , in particular rendall has shown global existence for small initial data in maximal - isotropic coordinates in . the previous results refer to massive particles but they do not immediately carry over to massless particles . this case is treated by dafermos in where global existence for small initial data is shown in double null coordinates . the spacetimes obtained in the studies [ 142 , 156 , 63 ] are all causally geodesically complete and appropriate decay rates of the metric and the matter quantities are given . in the case of small initial data the resulting spacetime is geodesically complete and no singularities form . a different scenario , which leads to a future geodesically complete spacetime , is to consider initial data where the particles are moving rapidly outwards . if the particles move sufficiently fast the matter disperses and the gravitational attraction is not strong enough to reverse the velocities of the particles to create a collapsing system . it is shown that the scenario described above can be realized , and that global existence holds . in section 3.7 we discuss results on the formation of black holes and trapped surfaces ; in particular , the results in will be presented . a corollary of the main result in concerns the issue of global existence and thus we mention it here . it is shown that a particular class of initial data , which lead to formation of black holes , have the property that the solutions exist for all schwarzschild time . the initial data consist of two parts : an inner part , which is a static solution of the einstein - vlasov system , and an outer part with matter moving inwards . the set - up is shown to preserve the direction of the momenta of the outer part of the matter , and it is also shown that in schwarzschild time the inner part and the outer part of the matter never interact in schwarzschild time . as was mentioned at the end of section 3.1 , the issue of global existence for general initial data is only relevant in certain time gauges since there are initial data leading to singular spacetimes . however , it is reasonable to believe that global existence for general data may hold in a polar time gauge or a maximal time gauge , cf . , and it is often conjectured in the literature that these time slicings are singularity avoiding . however , there is no proof of this statement for any matter model and it would be very satisfying to provide an answer to this conjecture for the einstein - vlasov system . a proof of global existence in these time coordinates would also be of great importance due to its relation to the weak cosmic censorship conjecture , cf . [ 61 , 62 , 65 ] . the methods of proofs in the cases described in sections 3.3 and 3.4 , where global existence has been shown , are all tailored to treat special classes of initial data and they will likely not apply in more general situations . in this section these results are all conditional in the sense that assumptions are made on the solutions , and not only on the initial data . the first study on global existence for general initial data is , which is carried out in schwarzschild coordinates . the authors introduce the following variables in the momentum space adapted to spherical symmetry , 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l : = \vert x{\vert ^2}\vert v{\vert ^2 } - { ( x \cdot v)^2},\;w = { { x \cdot v } \over r},$$\end{document } where l is the square of the angular momentum and w is the radial component of the momenta . a consequence of spherical symmetry is that angular momentum is conserved along the characteristics . in these variables the vlasov equation for f = f ( t , r , w , l ) becomes 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\partial _ t}w + { e^{\mu - \lambda}}{\mu _ r}e - { e^{\mu - \lambda}}{l \over { { r^3}e } } } \right){\partial _ w}f = 0,$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e = e(r , w , l ) = \sqrt { 1 + { w^2 } + l/{r^2}}.$$\end{document } the main result in shows that as long as there is no matter in the ball \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\{x \in { { \mathbb r}^3}:\vert x\vert \leq \epsilon \},$$\end{document } the estimate 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq { e^{\log q(0){e^{c(\epsilon)t}}}},$$\end{document } holds . here c( ) is a constant , which depends on . thus , in view of the continuation criterion this can be viewed as a global existence result outside the center of symmetry for initial data with compact support . this result rules out shell - crossing singularities , which are present when , e.g. , dust is used as a matter model . the bound of q is obtained by estimating each term individually in the characteristic equation associated with the vlasov equation ( 48 ) for the radial momentum . the einstein equations imply that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\mu _ r } = { m \over { { r^2}}}{e^{2\lambda } } + 4\pi rp{e^{2\lambda}}$$\end{document } where 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m(t,\,r ) = 4\pi \int\nolimits_0^r { { \eta ^2}\rho ( t,\eta ) d\eta,}$$\end{document } is the quasi local mass . thus , using ( 38 ) the characteristic equation consists of the two terms t1 = 4re , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${t_2 } = { e^{\mu + \lambda}}{m \over { { r^2}}}$\end{document } , together with a term , which is independent of the matter quantities . there is a distinct difference between the terms t1 and t2 due to the fact that m can be regarded as an average , since it is given as a space integral of the energy density , whereas j and p are point - wise terms . the method in makes use of a cancellation property of the radial momenta in t1 so that outside the center this term is manageable but in general it seems very unpleasant to have to treat point - wise terms of this kind . in rendall the bound on q(t ) is again obtained by estimating each term in the characteristic equation . in this case . however , the terms are , in analogy with the schwarzschild case , strongly singular at the center . in particular , the method avoids the point - wise terms by using the fact that the characteristic system can be written in a form such that green s formula in the plane can be applied . this results in a combination of terms involving second - order derivatives , which can be substituted for by one of the einstein equations . this method was first introduced in but the set - up is different in and the application of green s formula becomes very natural . in addition , the bound of q is improved compared to ( 49 ) and reads \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(t ) \leq q(0){e^{c(1 + t)/\epsilon t}}.$$\end{document } this bound is sufficient to conclude that global existence outside the center also holds for non - compact initial data . in addition to the global existence result outside the centre , it is shown in that as long as 3m(t , r ) r and j 0 , singularities can not form . note that in schwarzschild coordinates 2m(t , r ) r always , and that there are closed null geodesics if 3 m = r in the schwarzschild static spacetime . the method in also applies to the case of maximal - isotropic coordinates studied in . there is an improvement concerning the regularity of the terms that need to be estimated to obtain global existence in the general case . a consequence of is accordingly that the quite different proofs in and in are put on the same footing . we point out that the method can also be applied to the case of maximal - areal coordinates . the results discussed above concern time gauges , which are expected to be singularity avoiding so that the issue of global existence makes sense . an interpretation of these results is that first singularities ( where the notion of first is tied to the causal structure ) , in the non - trapped region , must emanate from the center and that this case has also been shown in double null - coordinates by dafermos and rendall in . the main motivation for studying the system in these coordinates has its origin from the method of proof of the cosmic - censorship conjecture for the einstein - scalar field system by christodoulou . an essential part of his method is based on the understanding of the formation of trapped surfaces . in it is shown that a single trapped surface or marginally - trapped surface in the maximal development implies that weak cosmic censorship holds the theorem holds true for any spherically - symmetric matter spacetime if the matter model is such that first singularities necessarily emanate from the center . the results in and in are not sufficient for concluding that the hypothesis of the matter needed in the theorem in is satisfied , since they concern a portion of the maximal development covered by particular coordinates . therefore , dafermos and rendall choose double - null coordinates , which cover the maximal development , and they show that the mentioned hypothesis is satisfied for vlasov matter . the main reason that the question of global existence in certain time coordinates discussed in the previous section 3.5 is of great importance is its relation to the cosmic censorship conjectures . now there is , in fact , no theorem in the literature , which guarantees that weak cosmic censorship follows from such a global existence result , but there are strong reasons to believe that this is the case , cf . and . hence , if initial data can be constructed , which lead to naked singularities , then either the conjecture that global existence holds generally is false or the viewpoint that global existence implies the absence of naked singularities is wrong . in view of a recent result by rendall and velazquez on self similar dust - like solutions for the massless einstein - vlasov system , this issue has much current interest . let us mention here that there is a previous study on self - similar solutions in the massless case by martn - garca and gundlach . however , this result is based on a scaling of the density function itself and therefore makes the result less related to the cauchy problem . also , their proof is , in part , based on numerics , which makes it harder to judge the relevance of the result . the main aim of the work is to establish self - similar solutions of the massive einstein - vlasov system and the present result can be viewed as a first step to achieving this . in the set - up , two simplifications are made . first , the authors study the massless case in order to find a scaling group , which leaves the system invariant . more precisely , the massless system is invariant under the scaling \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r \rightarrow \theta r,\;\;t \rightarrow \theta t,\;\;w \rightarrow { 1 \over { \sqrt \theta}}w,\;\;l \rightarrow \theta l.$$\end{document } the massless assumption seems not very restrictive since , if a singularity forms , the momenta will be large and therefore the influence of the rest mass of the particles will be negligible , so that asymptotically the solution can be self - similar also in the massive case , cf . the second simplification is that the possible radial momenta are restricted to two values , which means that the density function is a distribution in this variable . thus , the solutions can be thought of as intermediate between smooth solutions of the einstein - vlasov system and dust . for this simplified system it turns out that the existence question of self - similar solutions can be reduced to that of the existence of a certain type of solution of a four - dimensional system of ordinary differential equations depending on two parameters . the proof is based on a shooting argument and involves relating the dynamics of solutions of the four - dimensional system to that of solutions of certain two- and three - dimensional systems obtained from it by limiting processes . the reason that an ode system is obtained is due to the assumption on the radial momenta , and if regular initial data is considered , an ode system is not sufficient and a system of partial differential equations results . the solution is not asymptotically flat but there are ideas outlined in of how this can be overcome . it should be pointed out here that a similar problem occurs in the work by christodoulou for a scalar field , where the naked singularity solutions are obtained by truncating self - similar data . the singularity of the self - similar solution by rendall and velazquez is real in the sense that the kretschmann scalar curvature blows up . the asymptotic structure of the solution is striking in view of the conditional global existence result in . indeed , the self similar solution is such that j 0 , and 3m(t , r ) r asymptotically , but for any t , 3m(t , r ) > r for some t > t. in global existence follows if j 0 and if 3m(t , r ) r for all t. it is also the case that if m / r is close to 1/2 , then global existence holds in certain situations , cf . . hence , the asymptotic structure of the self - similar solution has properties , which have been shown to be difficult to treat in the search for a proof of global existence . we have previously mentioned that there exist initial data for the spherically - symmetric einstein - vlasov system , which lead to formation of black holes . the first result in this direction he shows that there exist initial data for the spherically - symmetric einstein - vlasov system such that a trapped surface forms in the evolution . the occurrence of a trapped surface signals the formation of an event horizon . as mentioned above , dafermos has proven that , if a spherically - symmetric spacetime contains a trapped surface and the matter model satisfies certain hypotheses , then weak cosmic censorship holds true . in it hence , by combining these results it follows that initial data exist , which lead to gravitational collapse and for which weak cosmic censorship holds . however , the proof in rests on a continuity argument , and it is not possible to tell whether or not a given initial data set will give rise to a black hole . this is in contrast to the result in , where explicit conditions on the initial data are given , which guarantee the formation of trapped surfaces in the evolution . the analysis is carried out in eddington - finkelstein coordinates and a central result in is to control the life span of the solution to ensure that there is sufficient time to form a trapped surface before the solution may break down . in particular , weak cosmic censorship holds for these initial data . in the formation of the event horizon in gravitational collapse one inner part and one outer part , in which all particles move inward initially . the reason for the inner part is that it is possible to choose the parameters for the data such that the particles of the outer matter part continue to move inward for all schwarzschild time as long as the particles do not interact with the inner part . this fact simplifies the analysis since the dynamics is much restricted when the particles keep the direction of their radial momenta . the main result is that explicit conditions on the initial data with adm mass m are given such that there is a family of outgoing null geodesics for which the area radius r along each geodesic is bounded by 2 m . it is furthermore shown that if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t \geq 0,\;{\rm{and}}\;r \geq 2 m + \alpha { e^{- \beta t}},$$\end{document } where and are positive constants , then f(t , r , , ) = 0 , and the metric equals the schwarzschild metric 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - ( 1 - { { 2 m } \over r})d{t^2 } + { ( 1 - { { 2 m } \over r})^{- 1}}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\phi ^2}),$$\end{document } representing a black hole with mass m. hence , spacetime converges asymptotically to the schwarzschild metric . the latter result does not reveal whether or not all the matter crosses r = 2 m or simply piles up at the event horizon . in it is shown that for initial data , which are closely related to those in , but such that the radial momenta are unbounded , all the matter do cross the event horizon asymptotically in schwarzschild time . this is in contrast to what happens to freely - falling observers in a static schwarzschild spacetime , since they will never reach the event horizon . the result in is reconsidered in , where an additional argument is given to match the definition of weak cosmic censorship given in . it is natural to relate the results of [ 20 , 24 ] to those of christodoulou on the spherically - symmetric einstein - scalar field system and . in it is shown that if the final bondi mass m is different from zero , the region exterior to the sphere r = 2 m tends to the schwarzschild metric with mass m similar to the result in . in explicit conditions on the initial data this paper played a crucial role in christodoulou s proof of the weak and strong cosmic censorship conjectures . the conditions on the initial data in allow the ratio of the hawking mass and the area radius to cover the full range , i.e. , 2m / r ( 0 , 1 ) , whereas the conditions in require 2m / r to be close to one . hence , it would be desirable to improve the conditions on the initial data in , although the conditions by christodoulou for a scalar field are not expected to be sufficient in the case of vlasov matter . in a numerical study on critical collapse for the einstein - vlasov system it has been shown by rein and rodewis that the numerical scheme has desirable convergence properties . ( in the vlasov - poisson case , convergence was proven in , see also ) . the speculation discussed above that there may be no naked singularities formed for any regular initial data is in part based on the fact that the naked singularities that occur in scalar field collapse appear to be associated with the existence of type ii critical collapse , while vlasov matter is of type i. the primary goal in was indeed to decide whether vlasov matter is type i or type ii . given small initial data , no black holes form and matter will disperse . for large data , black holes will form and consequently there is a transition regime separating dispersion of matter and formation of black holes . if we introduce a parameter a on the initial data such that for small a dispersion occurs and for large a a black hole is formed , we get a critical value ac separating these regions . if we take a > ac and denote by mb(a ) the mass of the black hole , then if mb(a ) 0 as a ac we have type ii matter , whereas for type i matter this limit is positive and there is a mass gap . for more information on critical collapse the conclusion of is that vlasov matter is of type i. there are two other independent numerical simulations on critical collapse for vlasov matter [ 128 , 21 ] . in these simulations , we end this section with a discussion of the spherically - symmetric einstein - vlasov - maxwell system , i.e. , the case considered above with charged particles . whereas the constraint equations in the uncharged case , written in schwarzschild coordinates , do not involve solving any difficulties once the distribution function is given , the charged case is more challenging . however , in it is shown that solutions to the constraint equations do exist for the einstein - vlasov - maxwell system . in local existence is shown together with a continuation criterion , and global existence for small initial data is shown in . in this section we discuss the einstein - vlasov system for cosmological spacetimes , i.e. , spacetimes that possess a compact cauchy surface . the particles in the kinetic description are in this case galaxies or even clusters of galaxies . the main goal is to determine the global properties of the solutions to the einstein - vlasov system for initial data given on a compact 3-manifold . in order to do so , a global time coordinate t must be found and the asymptotic behavior of the solutions when t tends to its limiting values has to be analyzed . this might correspond to approaching a singularity , e.g. , the big bang singularity , or to a phase of unending expansion . presently , the aim of most of the studies of the cosmological cauchy problem has been to show existence for unrestricted initial data and the results that have been obtained are in cases with symmetry ( see , however , , where to some extent global properties are shown in the case without symmetry ) . a recent and very extensive work by ringstrm has , on the other hand , a different aim , i.e. , to show stability of homogeneous cosmological models , and concerns the general case without symmetry . the size of the cauchy data is in this case very restricted but , since ringstrm allows general perturbations , there are no symmetries available to reduce the complexity of the einstein - vlasov system . the only spatially - homogeneous spacetimes admitting a compact cauchy surface are the bianchi types i , ix and the kantowski - sachs model ; to allow for cosmological solutions with more general symmetry types , it is enough to replace the condition that the spacetime is spatially homogeneous , with the condition that the universal covering of spacetime is spatially homogeneous . spacetimes with this property are called locally spatially homogeneous and these include , in addition , the bianchi types ii , iii , v , vi0 , vii0 , and viii . one of the first studies on the einstein - vlasov system for spatially - homogeneous spacetimes is the work by rendall . he chooses a gaussian time coordinate and investigates the maximal range of this time coordinate for solutions evolving from homogeneous data . for bianchi ix and for kantowski - sachs spacetimes he finds that the range is finite and that there is a curvature singularity in both the past and the future time directions . for the other bianchi types there is a curvature singularity in the past , and to the future spacetime is causally geodesically complete . although the questions on curvature singularities and geodesic completeness are very important , it is also desirable to have more detailed information on the asymptotic behavior of the solutions , and , in particular , to understand in which situations the choice of matter model is essential for the asymptotics . in recent years several studies on the einstein - vlasov system for spatially locally homogeneous spacetimes have been carried out with the goal to obtain a deeper understanding of the asymptotic structure of the solutions . roughly , these investigations can be divided into two cases : ( i ) studies on non - locally rotationally symmetric ( non - lrs ) bianchi i models and ( ii ) studies of lrs bianchi models . in case ( i ) rendall shows in that solutions converge to dust solutions for late times . under the additional assumption of small initial data this result is extended by nungesser , who gives the rate of convergence of the involved quantities . in rendall also raises the question of the existance of solutions with complicated oscillatory behavior towards the initial singularity may exist for vlasov matter , in contrast to perfect fluid matter . note that for a perfect fluid the pressure is isotropic , whereas for vlasov matter the pressure may be anisotropic , and this fact could be sufficient to drastically change the dynamics . this question is answered in , where the existence of a heteroclinic network is established as a possible asymptotic state . this implies a complicated oscillating behavior , which differs from the dynamics of perfect fluid solutions . the results in were then put in a more general context by calogero and heinzle , where quite general anisotropic matter models are considered . in case ( ii ) the asymptotic behaviour of solutions has been analyzed in [ 159 , 160 , 48 , 47 ] . in , the case of massless particles is considered , whereas the massive case is studied in . both the nature of the initial singularity and the phase of unlimited expansion are analyzed . the main concern in these two works is the behavior of bianchi models i , ii , and iii . a general conclusion is that the choice of matter model is very important since , for all symmetry classes studied , there are differences between the collision - less model and a perfect fluid model , both regarding the initial singularity and the expanding phase . the most striking example is for the bianchi ii models , where they find persistent oscillatory behavior near the singularity , which is quite different from the known behavior of bianchi type ii perfect fluid models . in it is also shown that solutions for massive particles are asymptotic to solutions with massless particles near the initial singularity . for bianchi i and ii , it is also proven that solutions with massive particles are asymptotic to dust solutions at late times . the investigation concerns a large class of anisotropic matter models , and , in particular , it is shown that solutions of the einstein - vlasov system with massless particles oscillate in the limit towards the past singularity for bianchi ix models . considers the homogeneous spacetimes with a cosmological constant for all bianchi models except bianchi type ix . she also obtains the time decay of the components of the energy momentum tensor as t , and she shows that spacetime is asymptotically dust - like . anguige studies the conformal einstein - vlasov system for massless particles , which admit an isotropic singularity . he shows that the cauchy problem is well posed with data consisting of the limiting density function at the singularity . in the spatially homogeneous case the metric can be written in a form that is independent of the spatial variables and this leads to an enormous simplification . another class of spacetimes that are highly symmetric but require the metric to be spatially dependent are those that admit a group of isometries acting on two - dimensional spacelike orbits , at least after passing to a covering manifold . the group may be two - dimensional ( local u(1 ) u(1 ) or t symmetry ) or three - dimensional ( spherical , plane , or hyperbolic symmetry ) . in all these cases , the quotient of spacetime by the symmetry group has the structure of a two - dimensional lorentzian manifold q. the orbits of the group action ( or appropriate quotients in the case of a local symmetry ) are called surfaces of symmetry . thus , there is a one - to - one correspondence between surfaces of symmetry and points of q. there is a major difference between the cases where the symmetry group is two- or three - dimensional . in the three - dimensional case no gravitational waves are admitted , in contrast to the two - dimensional case where the evolution part of the einstein equations are non - linear wave equations . three types of time coordinates that have been studied in the inhomogeneous case are cmc , areal , and conformal coordinates . a cmc time coordinate t is one where each hypersurface of constant time has constant mean curvature and on each hypersurface of this kind the value of t is the mean curvature of that slice . in the case of areal coordinates , the time coordinate is a function of the area of the surfaces of symmetry , e.g. , proportional to the area or proportional to the square root of the area . in the case of conformal coordinates , the advantage with a cmc approach is that the definition of a cmc hypersurface does not depend on any symmetry assumptions and it is possible that cmc foliations will exist for general spacetimes . the areal coordinate foliation , on the other hand , is adapted to the symmetry of spacetime but it has analytical advantages and detailed information about the asymptotics can be derived . the conformal coordinates have mainly served as a useful framework for the analysis to obtain geometrically - based time foliations . let us now consider spacetimes ( m , g ) admitting a three - dimensional group of isometries . the topology of m is assumed to be s f , with f a compact two - dimensional manifold . the universal covering \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\hat f}$\end{document } of f induces a spacetime \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\hat m,\hat g)$\end{document } by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat m = { \mathbb r } \times { s^1 } \times \hat f$\end{document } and = p*g , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } a three - dimensional group g of isometries is assumed to act on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\hat m,\hat g)$\end{document}. if f = s and g = so(3 ) , then ( m , g ) is called spherically symmetric ; if f = t and g = e2 ( euclidean group ) , then ( m , g ) is called plane symmetric ; and if f has genus greater than one and the connected component of the symmetry group g of the hyperbolic plane h acts isometrically on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\hat f = { h^2}$\end{document } , then ( m , g ) is said to have hyperbolic symmetry . in the case of spherical symmetry the existence of one compact cmc hypersurface implies that the whole spacetime can be covered by a cmc time coordinate that takes all real values [ 151 , 42 ] . the existence of one compact cmc hypersurface in this case was proven by henkel using the concept of prescribed mean curvature ( pmc ) foliation . accordingly , this gives a complete picture in the spherically symmetric case regarding cmc foliations . in the case of areal coordinates , rein has shown , under a size restriction on the initial data , that the past of an initial hyper - surface can be covered , and that the kretschmann scalar blows up . hence , the initial singularity for the restricted data is both a crushing and a curvature singularity . in the future direction it is shown that areal coordinates break down in finite time . in the case of plane and hyperbolic symmetry , global existence to the past was shown by rendall in cmc time . this implies that the past singularity is a crushing singularity since the mean curvature blows up at the singularity . also in these cases rein showed under a size restriction on the initial data , that global existence to the past in areal time and blow up of the kretschmann scalar curvature as the singularity is approached . hence , the singularity is both a crushing and a curvature singularity in these cases too . in both of these works the question of global existence to the future was left open . this gap was closed in , and global existence to the future was established in both cmc and areal time coordinates . the global existence result for cmc time is a consequence of the global existence theorem in areal coordinates , together with a theorem by henkel which shows that there exists at least one hypersurface with ( negative ) constant mean curvature . in addition , the past direction is analyzed in using areal coordinates , and global existence is shown without a size restriction on the data . it is not concluded if the past singularity , without the smallness condition on the data , is a curvature singularity as well . the issues discussed above have also been studied in the presence of a cosmological constant , cf . [ 182 , 184 ] . in particular it is shown that in the spherically - symmetric case , if < 0 , global existence to the future holds in areal time for some special classes of initial data , which is in contrast to the case with = 0 . in this context we also mention that surface symmetric spacetimes with vlasov matter and with a maxwell field have been investigated in . an interesting question , which essentially was left open in the studies mentioned above , is whether the areal time coordinate , which is positive by definition , takes all values in the range ( 0 , ) or only in ( r0 , ) for some positive r0 . it should here be pointed out that there is an example for vacuum spacetimes with t symmetry ( which includes the plane symmetric case ) where r0 > 0 . this question was first resolved by weaver for t symmetric spacetimes with vlasov matter . her result shows that if spacetime contains vlasov matter ( f 0 ) then r0 = 0 . smulevici has recently shown , under the same assumption , that r0 = 0 also in the hyperbolic case . smulevici also includes a cosmological constant and shows that both the results , for plane ( or t ) symmetry and hyperbolic symmetry , are valid for 0 . the important question of strong cosmic censorship for surface - symmetric spacetimes has recently been investigated by neat methods by dafermos and rendall [ 67 , 65 ] . the standard strategy to show cosmic censorship is to either show causal geodesic completeness in case there are no singularities , or to show that some curvature invariant blows up along any incomplete causal geodesic . in both cases no causal geodesic can leave the maximal cauchy development in any extension if we assume that the extension is c. in [ 67 , 65 ] two alternative approaches are investigated . the first method is independent of the matter model and exploits a rigidity property of cauchy horizons inherited from the killing fields . the areal time described above is defined in terms of the killing fields and a consequence of the method by dafermos and rendall is that the killing fields extend continuously to a cauchy horizon , if one exists . now , since global existence has been shown in areal time it follows that there can not be an extension of the maximal hyperbolic development to the future . the second method is dependent on vlasov matter and the idea is to follow the trajectory of a particle , which crosses the cauchy horizon and shows that the conservation laws for the particle motion associated with the symmetries of the spacetime , such as the angular momentum , lead to a contradiction . in most of the cases considered in there is an assumption on the initial data for the vlasov equation , which implies that the data have non - compact support in the momentum space the results of the studies [ 67 , 65 ] can be summarized as follows . for plane and hyperbolic symmetry strong cosmic censorship the restriction that matter has non - compact support in the momentum space is here imposed except in the plane case with = 0 . in the spherically - symmetric case cosmic censorship the difficulties to show cosmic censorship in this case are related to possible formation of extremal schwarzschild - de - sitter - type black holes . cosmic censorship in the past direction is also shown for all symmetry classes , and for all values of , for a special class of anti - trapped initial data . although the methods developed in [ 67 , 65 ] provide a lot of information on the asymptotic structure of the solutions , questions on geodesic completeness and curvature blow up are not answered . in a few cases , information on these issues has been obtained . as mentioned above , blow up of the kretschmann scalar curvature has been shown for restricted initial data . in the case of hyperbolic symmetry causal future geodesic completeness has been established by rein when the initial data are small . the authors show global existence to the future in areal time , and in particular they show that the spacetimes are future geodesically complete . it is shown that the de sitter solution acts as a model for the dynamics of the solutions by proving that the generalized kasner exponents tend to 1/3 as t , which in the plane case is the de sitter solution . the first study of spacetimes admitting a two - dimensional isometry group was carried out by rendall in the case of local t symmetry . for a discussion of the possible topologies of these spacetimes we refer to the original paper . in the model case the spacetime is topologically of the form t , and to simplify our discussion later on we write down the metric in areal coordinates for this type of spacetime : 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { g = { e^{2(\eta - u)}}(- \alpha d{t^2 } + d{\theta ^2 } ) + { e^{- 2u}}{t^2}{{[dy + h\;d\theta + m\;dt]}^2}}\\ { \quad \quad + { e^{2u}}{{[dx + a\;dy + ( g + ah)\;d\theta + ( l + am)dt]}^2}.}\\ \end{array}$$\end{document } here the metric coefficients , u , , a , h , l , and m depend on t and and , x , y s. in cmc coordinates are in fact considered rather than areal coordinates . under the hypothesis that there exists at least one cmc hypersurface , rendall proves for general initial data that the past of the given cmc hypersurface can be globally foliated by cmc hypersurfaces and that the mean curvature of these hypersurfaces blows up at the past singularity . the result in holds for vlasov matter and for matter described by a wave map . that the choice of matter model is important was shown in , where a non - global existence result for dust is given , which leads to examples of spacetimes that are not covered by a cmc foliation the plane case , where the symmetry group is three - dimensional , is one subcase and the form of the metric in areal coordinates is obtained by letting a = g = h = l = m = 0 and u = logt/2 in equation ( 52 ) . another subcase , which still admits only two killing fields ( and which includes plane symmetry as a special case ) , is gowdy symmetry . it is obtained by letting g = h = l = m = 0 in equation ( 52 ) . in gowdy symmetric spacetimes with vlasov matter are considered , and it is proven that the entire maximal globally hyperbolic spacetime can be foliated by constant areal time slices for general initial data . the areal coordinates are used in a direct way for showing global existence to the future , whereas the analysis for the past direction is carried out in conformal coordinates . these coordinates are not fixed to the geometry of spacetime and it is not clear that the entire past has been covered . a chain of geometrical arguments then shows that areal coordinates indeed cover the entire spacetime . the method in was in turn inspired by the work for vacuum spacetimes , where the idea of using conformal coordinates in the past direction was introduced . as pointed out in , the result by henkel guarantees the existence of one cmc hypersurface in the gowdy case and , together with the global areal foliation in , it follows that gowdy spacetimes with vlasov matter can be globally covered by cmc hypersurfaces as well . the more general case of t symmetry was considered in , where global cmc and areal time foliations were established for general initial data . in these results , the question whether or not the areal time coordinate takes values in ( 0 , ) or in ( r0 , ) , r0 > 0 , was left open . as we pointed out in section 4.2.1 , this issue was solved by weaver for t symmetric spacetimes with the conclusion that r0 = 0 , if the density function f is not identically zero initially . in the case of t symmetric spacetimes , with a positive cosmological constant , smulevici has shown global existence in areal time with the property that t ( 0 , ) . the issue of strong cosmic censorship for t symmetric spacetimes has been studied by dafermos and rendall using the methods , which were developed in the surface symmetric case described above . in strong cosmic censorship is shown under the same restriction on the initial data that was imposed in the surface symmetric case , which implies that the data have non - compact support in the momentum variable . their result has been extended to the case with a positive cosmological constant by smulevici . the present cosmological observations indicate that the expansion of the universe is accelerating , and this has influenced theoretical studies in the field during the last decade . one way to produce models with accelerated expansion is to choose a positive cosmological constant . another way is to include a non - linear scalar field among the matter fields , and in this section we review the results for the einstein - vlasov system , where a linear or non - linear scalar field have been included into the model . lee considers in the case where a non - linear scalar field is coupled to vlasov matter . the form of the energy momentum tensor then reads 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${t_{\alpha \beta } } = t_{\alpha \beta}^{{\rm{vlasov } } } + { \nabla _ \alpha}\phi { \nabla _ \beta}\phi - \left({{1 \over 2}{\nabla ^\gamma}\phi { \nabla _ \gamma}\phi + v(\phi ) } \right){g_{\alpha \beta}}.$$\end{document } here is the scalar field and v is a potential , and the bianchi identities lead to the following equation for the scalar field : 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\nabla ^\gamma}{\nabla _ \gamma}\phi = v^{\prime}(\phi){.}$$\end{document } under the assumption that v is a non - negative c function , global existence to the future is obtained , and if the potential is restricted to the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v(\phi ) = { v_0}{e^{- c\phi}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$0 < c < 4\sqrt \pi$\end{document } , then future geodesic completeness is proven . in the einstein - vlasov system with a linear scalar field is analyzed in the case of plane , spherical , and hyperbolic symmetry . here . a local existence theorem and a continuation criterion , involving bounds on derivatives of the scalar field in addition to a bound on the support of one of the moment variables , is proven . for the einstein scalar field system , i.e. , when f = 0 , the continuation criterion is shown to be satisfied in the future direction , and global existence follows in that case . the work extends the result in the plane and hyperbolic case to a global result in the future direction . in the plane case it is also shown that the singularity is crushing and that the kretschmann scalar diverges uniformly as the singularity is approached . in standard cosmology this is a strong assumption leading to severe restrictions of the possible geometries as well as of the topologies of the universe . thus , it is natural to ask if small perturbations of an initial data set , which corresponds to an expanding model of the standard type , give rise to solutions that are similar globally to the future ? in a recent work , ringstrm considers the einstein - vlasov system and he gives an affirmative answer to the stability question for some of the standard cosmologies . the standard model of the universe is spatially homogeneous and isotropic , has flat spatial hypersurfaces of homogeneity , a positive cosmological constant and the matter content consists of a radiation fluid and dust . hence , to investigate the question on stability it is natural to consider cosmological solutions with perfect fluid matter and a positive cosmological constant . however , as is shown by ringstrm , the standard model can be well approximated by a solution of the einstein - vlasov system with a positive cosmological constant . approximating dust with vlasov matter is straightforward , whereas approximating a radiation fluid is not . by choosing the initial support of the distribution function suitably , ringstrm shows that vlasov matter can be made to mimic a radiation fluid for a prescribed amount of time ; sooner or later the matter will behave like dust , but the time at which the approximation breaks down can be chosen to be large enough that the radiation is irrelevant to the future of that time in the standard picture . the main results in are stability of expanding , spatially compact , spatially locally homogeneous solutions to the einstein - vlasov system with a positive cosmological constant as well as a construction of solutions with arbitrary compact spatial topology . in other words , the assumption of almost spatial homogeneity and isotropy does not seem to impose a restriction on the allowed spatial topologies . let us mention here some related works although these do not concern the einstein - vlasov system . ringstrm considers the case where the matter model is a non - linear scalar field in and . the background solutions , which ringstrm perturb and which are shown to be stable , have accelerated expansion . in the expansion the corresponding problem for a fluid has been treated in and , and the newtonian case is investigated in and for vlasov and fluid matter respectively . equilibrium states in galactic dynamics can be described as static or stationary solutions of the einstein - vlasov system , or of the vlasov - poisson system in the newtonian case . here we consider the relativistic case and we refer to the excellent review paper for the newtonian case . first , we discuss spherically - symmetric solutions for which the structure is quite well understood . on the other hand , almost nothing is known about the stability of the spherically - symmetric static solutions of the einstein - vlasov system , which is in sharp contrast to the situation for the vlasov - poisson system . at the end of this section let the metric have the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - { e^{2\mu ( r)}}d{t^2 } + { e^{2\lambda ( r)}}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\varphi ^2}),$$\end{document } where r 0 , [ 0 , ] , [ 0 , 2 ] . as before , asymptotic flatness is expressed by the boundary conditions \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset{r \rightarrow \infty}{\lim } \lambda ( r ) = \underset{r \rightarrow \infty}{\lim } \mu ( r ) = 0,$$\end{document } and a regular center requires \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\lambda ( 0 ) = 0.$$\end{document } following the notation in section 3.1 , the time - independent einstein - vlasov system reads 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{\mu - \lambda}}{v \over { \sqrt { 1 + \vert v{\vert ^2 } } } } \cdot { \nabla _ x}f - \sqrt { 1 + \vert v{\vert ^2 } } { e^{\mu - \lambda}}{\mu _ r}{x \over r } \cdot { \nabla _ v}f = 0,$$\end{document } 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\lambda _ r } - 1 ) + 1 = 8\pi { r^2}\rho,$$\end{document } 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^{- 2\lambda}}(2r{\mu _ r } + 1 ) - 1 = 8\pi { r^2}p.$$\end{document } recall that there is an additional equation ( 39 ) of second order , which contains the tangential pressure pt , but we leave it out since it follows from the equations above . the matter quantities are defined as before : 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\rho ( x ) = \int\nolimits_{{{\mathbb r}^3 } } { \sqrt { 1 + \vert v{\vert ^2 } } f(x , v)dv,}$$\end{document } 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(x ) = \int\nolimits_{{{\mathbb r}^3 } } { { { \left({{{x \cdot v } \over r } } \right)}^2}f(x , v){{dv } \over { \sqrt { 1 + \vert v{\vert ^2}}}}.}$$\end{document } the quantities \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$e : = { e^{\mu ( r)}}\sqrt { 1 + \vert v{\vert ^2 } } , \quad l = \vert x{\vert ^2}\vert v{\vert ^2 } - { ( x \cdot v)^2 } = \vert x \times v{\vert ^2},$$\end{document } are conserved along characteristics . if we let 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(x , v ) = \phi ( e , l),$$\end{document } for some function , the vlasov equation is automatically satisfied . a common assumption in the literature is to restrict the form of to 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi ( e , l ) = \phi ( e)(l - { l_0 } ) _ + ^l,$$\end{document } where l > 1/2 , l0 0 and x+ = max{x , 0}. if we furthermore assume that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi ( e ) = ( e - { e_0 } ) _ + ^k,\;k > - 1,$$\end{document } for some positive constant e0 , then we obtain the polytropic ansatz . the case of isotropic pressure is obtained by letting l = 0 so that only depends on e. in passing , we mention that for the vlasov - poisson system it has been shown that every static spherically - symmetric solution must have the form f = (e , l ) . this is referred to as jeans theorem . it was an open question for some time whether or not this was also true for the einstein - vlasov system . this was settled in 1999 by schaeffer , who found solutions that do not have this particular form globally on phase space , and consequently , jeans theorem is not valid in the relativistic case . however , almost all results on static solutions are based on this ansatz . by inserting the ansatz f(x , v ) = (e , l ) in the matter quantities and p , a non - linear system for and is obtained , in which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { e^{- 2\lambda}}(2r{\lambda _ r } - 1 ) + 1 = 8\pi { r^2}{g_\phi}(r,\mu),}\\ { { e^{- 2\lambda}}(2r{\mu _ r } + 1 ) - 1 = 8\pi { r^2}{h_\phi}(r,\mu),}\\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { g_\phi}(r,\mu ) = { { 2\pi } \over { { r^2}}}\int\nolimits_1^\infty { \int\nolimits_0^{{r^2}({\epsilon ^2 } - 1 ) } { \phi ( { e^{\mu ( r)}}\epsilon , l){{{\epsilon ^2 } } \over { \sqrt { { \epsilon ^2 } - 1 - l/{r^2}}}}dld\epsilon}}}\\ { { h_\phi}(r,\mu ) = { { 2\pi } \over { { r^2}}}\int\nolimits_1^\infty { \int\nolimits_0^{{r^2}({\epsilon ^2 } - 1 ) } { \phi ( { e^{\mu ( r)}}\epsilon , l)\sqrt { { \epsilon ^2 } - 1 - l/{r^2 } } dld\epsilon.}}}\\ \end{array}$$\end{document } existence of solutions to this system was first proven in the case of isotropic pressure in , and extended to anisotropic pressure in . the main difficulty is to show that the solutions have finite adm mass and compact support . the argument in these works to obtain a solution of compact support is to perturb a steady state of the vlasov - poisson system , which is known to have compact support . two different types of solutions are constructed , those with a regular centre [ 144 , 134 ] , and those with a schwarzschild singularity in the centre . in , rein and rendall go beyond the polytropic ansatz and obtain steady states with compact support and finite mass under the assumption that satisfies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\phi ( e , l ) = c(e - { e_0 } ) _ + ^{\delta + k}){l^l}\;{\rm{as}}\;e \rightarrow { e_0},$$\end{document } where k > 1 , l > 1/2 , k + l + 1/2 > 0 , k < l + 3/2 . this result is obtained in a more direct way and is not based on the perturbation argument used in [ 144 , 134 ] . their method is inspired by a work on stellar models by makino , in which he considers steady states of the euler - einstein system . in there is also a discussion about steady states that appear in the astrophysics literature , and it is shown that their result applies to most of these steady states . an alternative method to obtain steady states with finite radius and finite mass , which is based on a dynamical system analysis , all solutions described so far have the property that the support of contains a ball about the centre . in rein showed that steady states also exist whose support is a finite , spherically - symmetric shell with a vacuum region in the center . in it a systematic study of the structure of spherically - symmetric static solutions was carried out mainly by numerical means in and we now present the conclusions of this investigation . by prescribing the value (0 ) , the equations can be solved , but the resulting solution will in general not satisfy the boundary condition ( ) = 0 , but it will have some finite limit . it is then possible to shift both the cut - off energy e0 and the solution by this limit to obtain a solution , which satisfies ( ) = 0 . a convenient way to handle the problem that e0 and (0 ) can not both be treated as free parameters is to use the ansatz \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(x , v ) = ( 1 - e/{e_0 } ) _ + ^l , k \geq 0,\;l > - 1/2,k < 3l + 7/2,$$\end{document } as in . this gives an equation for , which can be rewritten in terms of the function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$y(r ) = { { { e^{\mu ( r ) } } } \over { { e_0}}}.$$\end{document } in this way the cut - of energy disappears as a free parameter of the problem and we thus have the four free parameters k , l , l0 and y(0 ) . the structure of the static solutions obtained in is as follows : if l0 = 0 the energy density can be strictly positive or vanish at r = 0 ( depending on l ) but it is always strictly positive sufficiently close to r = 0 . hence , the support of the matter is an interval [ 0 , r1 ] with r1 > 0 , and we call such states ball configurations . if l0 > 0 the support is in an interval [ r0 , r1 ] , r0 > 0 , and we call such steady states for shells . the value y(0 ) determines how compact or relativistic the steady state is , and the smaller values the more relativistic . for large values , recall y(0 ) 1/e0 , a pure shell or a pure ball configuration is obtained , cf . note that we depict the behavior of but we remark that the pressure terms behave similarly but the amplitudes of p and pt can be very different , i.e. , the steady states can be highly anisotropic . figure 1a pure shell for moderate values of y(0 ) the solutions have a distinct inner peak and a tail - like outer peak , and by making y(0 ) smaller more peaks appear , cf . figure 2multi - peaks of ball configurations , l0 = 0 multi - peaks of ball configurations , l0 = 0 in the case of shells there is a similar structure but in this case the peaks can either be separated by vacuum regions or by thin atmospheric regions as in the case of ball configurations . an example with multi - peaks , where some of the peaks are separated by vacuum regions , is given in figure 3 . figure 3multi - peaks of a shell multi - peaks of a shell a different feature of the structure of static solutions is the issue of spirals . for a fixed ansatz of the density function f , there is a one - parameter family of static solutions , which are parameterized by y(0 ) . a natural question to ask is how the adm mass m and the radius of the support r change along such a family . by plotting for each y(0 ) the resulting values for r and m a curve is obtained , which reflects how radius and mass are related along such a one - parameter family of steady states . figure 4 . it is shown in that in the isotropic case , where l = l0 = 0 the radius - mass curves always have a spiral form . figure 4(r , m ) spiral for k = 0 , l = 10.5 , lo = 0 , 0.01 y(0 ) 0.99 ( r , m ) spiral for k = 0 , l = 10.5 , lo = 0 , 0.01 y(0 ) 0.99 another aspect of the structure of steady states investigated numerically in concerns the buchdahl inequality . if a steady state has support in [ r0 , r1 ] , then the adm mass m of the configuration is m = m(r1 ) , where the quasi local mass m(r ) is given by equation ( 50 ) in schwarzschild coordinates . in view of the schwarzschild metric ( 51 ) , schwarzschild asked already in 1916 the question : how large can 2m / r possibly be ? he gave the answer 2m / r 8/9 in the special case of the schwarzschild interior solution , which has constant energy density and isotropic pressure . in 1959 buchdahl extended his result to isotropic solutions for which the energy density is non - increasing outwards and he showed that also in this case 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{2 m } \over r } \leq { 8 \over 9}.$$\end{document } this is sometimes called the buchdahl inequality . let us remark that the buchdahl inequality can obviously be written as m / r 4/9 , but since it is the quantity 2m / r , which appears in the schwarzschild metric ( 51 ) , it is common to keep the form of equation ( 62 ) . a bound on 2m / r has an immediate observational consequence since it limits the possible gravitational red shift of a spherically - symmetric static object . in particular , the overwhelming number of the steady states of the einstein - vlasov system have neither an isotropic pressure nor a non - increasing energy density , but nevertheless 2m / r is always found to be less than 8/9 in the numerical study . also for other matter models as pointed out by guven and murchadha , neither of the buchdahl assumptions hold in a simple soap bubble and they do not approximate any known topologically stable field configuration . in addition , there are also several astrophysical models of stars , which are anisotropic . lemaitre proposed a model of an anisotropic star already in 1933 , and binney and tremaine explicitly allow for an anisotropy coefficient . hence , it is an important question to investigate bounds on 2m / r under less restrictive assumptions . in it is shown that for any static solution of the spherically - symmetric einstein equation , not necessarily of the einstein - vlasov system , for which p 0 , and 63\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p + 2{p_t } \leq \omega \rho,$$\end{document } the following inequality holds 64\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{2m(r ) } \over r } \leq { { { { ( 1 + 2\omega)}^2 } - 1 } \over { { { ( 1 + 2\omega)}^2}}}.$$\end{document } moreover , the inequality is sharp and sharpness is obtained uniquely by an infinitely thin shell solution . note in particular that for vlasov matter = 1 and that the right - hand side then equals 8/9 as in the buchdahl inequality . an alternative proof was given in and their method applies to a larger class of conditions on , p and pt than the one given in equation ( 63 ) . on the other hand , the result in is weaker than the result in in the sense that the latter method implies that the steady state that saturates the inequality is unique ; it is an infinitely thin shell . the studies [ 10 , 103 ] are of general character and in particular it is not shown that solutions exist to the coupled einstein - matter system , which can saturate the inequality . for instance , it is natural to ask if there are solutions of the einstein - vlasov system , which have 2m / r arbitrarily close to 8/9 . this question is given an affirmative answer in , where in particular it is shown that arbitrarily thin shells exist , which are regular solutions of the spherically - symmetric einstein - vlasov system . using the strategy in it follows that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset{r}{\sup}{{2m(r ) } \over r } \rightarrow { 8 \over 9},$$\end{document } in the limit when the shells become infinitely thin . the question of finding an upper bound on 2m / r can be extended to charged objects and to the case with a positive cosmological constant . the spacetime outside a spherically - symmetric charged object is given by the reissner - nordstrm metric \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - ( 1 - { { 2 m } \over r } + { q \over { { r^2}}})d{t^2 } + { ( 1 - { { 2 m } \over r } + { q \over { { r^2}}})^{- 1}}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta d{\phi ^2}),$$\end{document } where q is the total charge of the object . the quantity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$1 - { { 2 m } \over r } + { q \over { { r^2}}}$\end{document } is zero when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r _ \pm } = m \pm \sqrt { { m^2 } - { q^2}}$\end{document } , and r is called the inner and outer horizon respectively of a reissner - nordstrm black hole . a buchdahl type inequality gives a lower bound of the area radius of a static object and this radius is thus often called the critical stability radius . it is shown in that a spherically - symmetric static solution of the einstein - maxwell system for which p 0 , p + 2pt , and q < m satisfy 65\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\sqrt m \leq { { \sqrt r } \over 3 } + \sqrt { { r \over 9 } + { { { q^2 } } \over { 3r}}}.$$\end{document } note , in particular , that the inequality holds for solutions of the einstein - vlasov - maxwell system , since the conditions above are always satisfied in this case . this inequality ( 65 ) implies that the stability radius is outside the outer horizon of a reissner - nordstrm black hole . in the relevance of an inequality of this kind on aspects in black - hole physics an infinitely thin shell solution does saturate the inequality ( 65 ) , but numerical evidence is given in that there is also another type of solution , which saturates the inequality for which the inner and outer horizon coincide . the study in is concerned with the non - charged situation when a positive cosmological constant is included . the following inequality is derived \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m \over r } \leq { 2 \over 9 } - { { \lambda { r^2 } } \over 3 } + { 2 \over 9}\sqrt { 1 + 3\lambda { r^2}},$$\end{document } for solutions for which p 0 , p + 2pt , and 0 r 1 . in this situation , an infinitely thin shell solution does not generally saturate the inequality but does so in the two degenerate situations r = 0 and r = 1 . in the latter case there is a constant density solution , and the exterior spacetime is the nariai solution , which saturates the inequality and the saturating solution is thus non - unique . in this case , the cosmological horizon and the black hole horizon coincide , which is in analogy with the charged situation described above where the inner and outer horizons coincide when uniqueness is likely lost . an important problem is the question of the stability of spherically - symmetric steady states . at present , there are almost no theoretical results on the stability of the steady states of the einstein - vlasov system . wolansky has applied the energy - casimir method and obtained some insights , but the theory is much less developed than in the vlasov - poisson case and the stability problem is essentially open . the situation is very different for the vlasov - poisson system , and we refer to for a review on the results in this case . however , there are numerical studies [ 21 , 100 , 171 ] on the stability of spherically - symmetric steady states for the einstein - vlasov system . the latter two studies concern isotropic steady states , whereas the first , in addition , treats anisotropic steady states . here we present the conclusions of , emphasizing that these agree with the conclusions in [ 171 , 100 ] for isotropic states . to allow for trapped surfaces , maximal - areal coordinates are used , i.e. , the metric is written in the following form in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - ( { \alpha ^2 } - { a^2}{\beta ^2})d{t^2 } + 2{a^2}\beta dtdr + { a^2}d{r^2 } + { r^2}(d{\theta ^2 } + { \sin ^2}\theta \,d{\phi ^2}).$$\end{document } here the metric coefficients , , and a depend on t and r 0 , and a are positive , and the polar angles [ 0 , ] and [ 0 , 2 ] parameterize the unit sphere . a maximal gauge condition is then imposed , which means that each hypersurface of constant t has vanishing mean curvature . the boundary conditions , which guarantee asymptotic flatness and a regular center , are given by 66\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a(t,0 ) = a(t,\infty ) = \alpha ( t,\infty ) = 1.$$\end{document } steady states are numerically constructed , and these are then perturbed in order to investigate the stability . more precisely , to construct the steady states the polytropic ansatz is used , cf . section 5.1 , 67\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(r , w , l ) = \phi ( e , l ) = ( { e_0 } - e ) _ + ^l.$$\end{document } by specifying values on e0 , l0 and (0 ) steady states are numerically constructed . the distribution function fs of the steady state is then multiplied by an amplitude a , so that a new , perturbed distribution function is obtained . we remark that also other types of perturbations are analyzed in . for k and l fixed each steady state is characterized by its central red shift zc and its fractional binding energy eb , which are defined by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${z_c } = { 1 \over { \alpha ( 0 ) } } - 1,\;{e_b } = { { { e_b } } \over { { m_0}}},\;{\rm{where}}\;{e_b } = { m_0 } - m.$$\end{document } here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_0 } = 4{\pi ^2}\int\nolimits_0^\infty { \int\nolimits_{- \infty}^\infty { \int\nolimits_0^\infty { a(t , r)f(t , r , w , l ) } dl\;dw\;dr}}$$\end{document } is the total number of particles , which , since all particles have rest mass one , equals the rest mass of the system . m is the adm mass given by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$m = \int\nolimits_0^\infty { \left({4\pi \rho ( t , r ) + { 3 \over 2}{\kappa ^2}(t , r ) } \right){r^2}dr,}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$k = \beta / r\alpha$\end{document } both m0 and m are conserved quantities . the central redshift is the redshift of a photon emitted from the center and received at infinity , and the binding energy eb is the difference of the rest mass and the adm mass . in figure 5 and figure 6 the relation between the fractional binding energy and the central redshift is given for two different cases . figure 5k = 0 , l = 0 , l0 = 0.1 figure 6k = 0 , l = 3/2 , l0 = 0.1 k = 0 , l = 0 , l0 = 0.1 k = 0 , l = 3/2 , l0 = 0.1 the relevance of these concepts for the stability properties of steady states was first discussed by zeldovich and podurets , who argued that it should be possible to diagnose the stability from binding energy considerations . zeldovich and novikov then conjectured that the binding energy maximum along a steady state sequence signals the onset of instability . varying the parameters k , l and l0 give rise to essentially the same tables , cf . . table 1k = 0 and l = 1/2 . z c e b a < 1a > 10.210.032stablestable0.340.040stablestable0.390.040stablestable0.420.041stableunstable0.460.040stableunstable0.560.036stableunstable0.650.029stableunstable0.820.008stableunstable0.950.015unstableunstable1.200.078unstableunstable if we first consider perturbations with a > 1 , it is found that steady states with small values on zc ( less than approximately 0.40 in this case ) are stable , i.e. , the perturbed solutions stay in a neighbourhood of the static solution . a careful investigation of the perturbed solutions indicates that they oscillate in a periodic way . for larger values of zc hence , for perturbations with a > 1 the value of zc alone seems to determine the stability features of the steady states . plotting eb versus zc with higher resolution , cf . , gives support to the conjecture by novikov and zeldovich mentioned above that the maximum of eb along a sequence of steady states signals the onset of instability . consider a steady state with eb > 0 and a perturbation with a < 1 but close to 1 so that the fractional binding energy remains positive . the perturbed solution then drifts outwards , turns back and reimplodes , and comes close to its initial state , and then continues to expand and reimplode and thus oscillates , cf . figure 7zc = 0.47 , eb = 0.04 , a = 0.99 , t = 90.0 zc = 0.47 , eb = 0.04 , a = 0.99 , t = 90.0 in it is stated ( without proof ) that if eb > 0 the solution must ultimately reimplode and the simulations in support that it is true . for negative values of eb , the solutions with a < 1 disperse to infinity . a simple analytic argument is given in , which relates the question , whether a solution disperses or not . it is shown that if a shell solution has an expanding vacuum region of radius r(t ) at the center with r(t ) for t , i.e. , the solution disperses in a strong sense , then necessarily m0 m , i.e. , eb 0 . as we have seen above , a broad variety of static solutions of the einstein - vlasov system has been established , all of which share the restriction that they are spherically symmetric . the recent investigation removes this restriction and proves the existence of static solutions of the einstein - vlasov system , which are axially symmetric but not spherically symmetric . from the applications point of view this symmetry assumption is more realistic than spherical symmetry , and from the mathematics point of view the complexity of the einstein field equations increases drastically if one gives up spherical symmetry . before discussing this result , let us mention that similar results have been obtained for two other matter models . in the case of a perfect fluid , heilig showed the existence of axisymmetric stationary solutions in . these solutions have non - zero angular momentum since static solutions are necessarily spherically symmetric . in this respect the existence of static axisymmetric solutions of elastic matter , which are not spherically symmetric , was proven in . let us now briefly discuss the method of proof in , which relies on an application of the implicit function theorem . also , the proofs in [ 92 , 1 , 2 ] make use of the implicit function theorem , but apart from this fact the methods are quite different . the set - up of the problem in follows the work of bardeen , where the metric is written in the form 68\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d{s^2 } = - { c^2}{e^{2\nu/{c^2}}}d{t^2 } + { e^{2\mu}}d{\rho ^2 } + { e^{2\mu}}d{z^2 } + { \rho ^2}{b^2}{e^{- 2\nu/{c^2}}}d{\varphi ^2}$$\end{document } for functions , b , depending on and z , where t , [ 0 , [ , z and [ 0 , 2 ] . the killing vector fields t and correspond to the stationarity and axial symmetry of the spacetime . solutions are obtained by perturbing off spherically symmetric steady states of the vlasov - poisson system via the implicit function theorem and the reason for writing /c in the metric , instead of , is that converges to the newtonian potential un of the steady state in the limit c . asymptotic flatness is expressed by the boundary conditions 69\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\underset{\vert ( \rho , z)\vert \rightarrow \infty}{\lim}\nu ( \rho , z ) = \underset{\vert ( \rho , z)\vert \rightarrow \infty}{\lim } \mu ( \rho , z ) = 0,\;\;\underset{\vert ( \rho , z)\vert \rightarrow \infty}{\lim } b(\rho , z ) = 1.$$\end{document } in addition the solutions are required to be locally flat at the axis of symmetry , which implies the condition 70\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\nu ( 0,z)/{c^2 } + \mu ( 0,z ) = \ln b(0,z),\;z \in { \mathbb r}.$$\end{document } let us now recall from section 5.1 the strategy to construct static solutions by using an ansatz of the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(x , v ) = \phi ( e , l),$$\end{document } where e and l are conserved quantities along characteristics . due to the symmetries of the metric ( 68 ) the following quantities are constant along geodesics : 71\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { e : = - g(\partial/\partial t,{p^a } ) = { c^2}{e^{2\nu/{c^2}}}{p^0}}\\ { \quad \ ; = { c^2}{e^{\nu/{c^2}}}\sqrt { 1 + { c^{- 2}}({e^{2\mu}}{{({p^1})}^2 } + { e^{2\mu}}{{({p^2})}^2 } + { \rho ^2}{b^2}{e^{- 2\nu/{c^2}}}{{({p^3})}^2})},}\\ \end{array}$$\end{document } 72\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$l : = - g(\partial/\partial \varphi , { p^a } ) = { \rho ^2}{b^2}{e^{- 2\nu/{c^2}}}{p^3}.$$\end{document } here p are the canonical momenta . e can be thought of as a local or particle energy and l is the angular momentum of a particle with respect to the axis of symmetry . for a sufficiently regular the ansatz function f satisfies the vlasov equation and upon insertion of this ansatz into the definition of the energy momentum tensor ( 32 ) the latter becomes a functional ta = t( , b , ) of the unknown metric functions , b , . it then remains to solve the einstein equations with this energy momentum tensor as right - hand side . the newtonian limit of the einstein - vlasov system is the vlasov - poisson system and the strategy in is to perturb off spherically symmetric steady states of the vlasov - poisson system via the implicit function theorem to obtain axisymmetric solutions . indeed , the main result of specifies conditions on the ansatz function such that a two parameter ( and ) family of axially - symmetric solutions of the einstein - vlasov system passes through the corresponding spherically symmetric , newtonian steady state , whose ansatz function we denote by . the parameter = 1/c turns on general relativity and the parameter turns on the dependence on l. since l is not invariant under arbitrary rotations about the origin the solution is not spherically symmetric if f depends on l. it should also be mentioned that although is a priori small , which means that c is large , the scaling symmetry of the einstein - vlasov system can be used to obtain solutions corresponding to the physically correct value of c. the most striking condition on the ansatz function , or rather on the ansatz function of the corresponding vlasov - poisson system , needed to carry out the proof is that it must satisfy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$6 + 4\pi { r^2}{a_n}(r ) > 0,\quad r \in [ 0,\infty [ , $ $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${a_n}(r ) : = \int\nolimits_{{{\mathbb r}^3 } } { \phi ^{\prime } } \left({{1 \over 2}\vert v{\vert ^2 } + { u_n}(r ) } \right)dv.$$\end{document } an important argument in the proof is indeed to justify that there are steady states of the vlasov - poisson system satisfying this condition . it is of course desirable to extend the result in to stationary solutions with rotation . moreover , the deviation from spherically symmetry of the solutions in is small and an interesting open question is the existence of disk - like models of galaxies . in the vlasov - poisson case this has been shown in . there are at least 40 types of legionella bacteria , half of which are capable of producing disease in humans . the legionella pneumophila bacterium we describe the case of a 60-year - old woman with a history of diabetes mellitus and arterial hypertension who was admitted to our hospital with fever and symptoms of respiratory infection , diarrhea , and acute renal failure . we used real - time polymerase chain reaction ( pcr ) to detect l. pneumophila dna in peripheral blood and serum samples and urine antigen from a patient with pneumonia . since legionella is a cause of 2% to 15% of all community - acquired pneumonias that require hospitalization , legionellosis should be taken into account in an atypical pulmonary infection and not be forgotten . moreover , real - time pcr should be considered a useful diagnostic method . legionella species are gram - negative bacteria that are ubiquitous in both natural aquatic and moist soil and muddy environments and in artificial aquatic habitats.1,2 human infection with legionella spp . has two distinct forms : legionnaires disease , a more severe form of infection which includes pneumonia , and pontiac fever , a milder febrile flu - like illness without pneumonia.3 legionella stands as the cause of community- acquired pneumonia ( cap ) in 2%15% of all caps that require hospitalization . the clinical and radiological features of legionella pneumonia are nonspecific , and the diagnosis depends on laboratory tests.4 this paper reports a case of pneumonia caused by legionella pneumophila that was admitted to the general hospital of komotini in greece . a 60-year - old female with a history of diabetes mellitus , chronic obstructive pulmonary disease , and arterial hypertension with a 4-day history of watery diarrhea and temperature was admitted to our hospital . the findings from physical examinations on admission were as follows : temperature 38.6c , blood pressure 130/95 mm hg , heart rate 117 beats / min , and respiratory rate 28 breaths / min . laboratory findings on admission were as follows : white blood cell ( wbc ) count was 14,280/l , creatinine was 3.4 mg / dl , the erythrocyte sedimentation rate ( esr ) was 110/h , procacitonin ( pct ) 2 ng / ml , c - reactive protein ( crp ) 14 mg / dl ( table 1 ) . we considered the elevated creatinine levels as acute , because at her previous laboratory tests her creatinine values never surpassed 1.3 mg / dl . arterial blood gas showed hypoxemia , as partial pressure of oxygen on air was 56 mm hg.5 the chest x - ray showed infiltration of the left lung ( figure 1 ) . with a clinical diagnosis of pneumonia , urine samples for l. pneumophila and streptococcus pneumoniae antigen , gram stain sputum , and blood specimens upon admission were collected from the patient for culture testing.6 the results were reported negative . the pneumonia severity index ( psi ) was evaluated as class 3 with a mortality rate of 0.9%.7 the patient was treated empirically with 1 g amoxycillin / clavulanic acid three times daily and 500 mg clarithromycin two times daily for 2 days . empiric antibiotic treatment was added upon admission based on elevated values of crp , wbc , esr , and chest x - ray findings , since early antibiotic treatment prevents progression of the disease and these markers are known to be elevated in infectious diseases.8,9 the clinical status of the patient was deteriorating , and there was a marked progression of the infiltrates on the chest x - ray ; the left infiltrate progressed to bilateral shadows . the laboratory values were as follows on the 2nd hospital day : wbc count 17,780/l , crp 18 mg / dl , pct 2 ng / ml , and esr 120/h . the patient s hypoxemia increased : partial pressure of oxygen in arterial blood ( pao2 ) 47 mmhg , partial pressure of carbon dioxide in arterial blood ( paco2 ) 25.4 mm hg , and ph 7.5 ( table 1 ) . one day after her admission to hospital ( day 2 ) ( figure 2 ) , two blood samples , one serum sample and a second urine sample were taken and tested for l. pneumophila with a real - time polymerase chain reaction ( pcr ) kit ( aqua screen l. pneumophila - detection kit for real - time pcr ; minerva biolabs , minerva , oh ) and with culture method . a portion of 200 l of blood and an equal volume of serum were plated on buffered charcoal yeast extract ( bcye ) ( oxoid , reading , uk ) with l - cysteine , on bcye without l - cysteine , and on gvpc ( gas vesicle protein c ) . the antibiotics were immediately changed to levofloxacin after the positive real - time pcr and legionella urinary antigen detection test . the patient responded after therapy with levofloxacin , on day four ( figure 3 ) . her general condition , as well as radiographic and laboratory findings , gradually improved and the patient was discharged on the 21st day of hospitalization ( figure 4 ) . to identify the source of the infection , the patient s relatives were interviewed . exposure histories revealed that the patient had travelled abroad and visited an operating spa pool 2 weeks before the day of onset . after she returned , she remained in her village until her admission to hospital . as there were no cooling towers or any aerosol - generating systems in an area of 2 km from the house , and there was no access to the spa pool abroad , environmental samples were taken only from the home water supplies , and standard methods were processed . the incidence of legionnaires disease has increased in the last decade since the introduction of urinary antigen immunoassays.10,11 this test accounts for most of the diagnostics due to its high sensitivity and ease of use.12 l. pneumophila has become one of the leading causes of cap in adults , accounting for 6%14% of cases requiring hospitalization in recent studies.13,14 legionnaires disease occurs sporadically and in outbreaks , with the sporadic form representing 65%82% of the cases.10,11,15 nevertheless , the number of confirmed community outbreaks including more than 100 cases has increased in recent years due to the use of legionella antigenuria.11,15 routine testing for legionella urinary antigen has increased the number of diagnostics of legionnaires disease and has allowed earlier diagnosis and treatment , greatly improving the prognosis.16 this has been particularly true for milder cases , mainly in the outbreak setting.17 however , most of the knowledge on risk factors , clinical presentation , and outcome of community - acquired legionnaires disease is based on studies performed before routine urinary antigen testing was adopted.18,19 moreover , recent community outbreaks have contributed to the better understanding of legionnaires disease in this setting.2022 legionella pneumophila has been recognized as an important cause of both cap and nosocomial pneumonia.1,10,11,23 environmental systems , such as air conditioning cooling towers , evaporative condensers , whirlpools , and hot spring baths have hosted and transmitted the organism . cases of legionella pneumonia presumably transmitted from contaminated hot spring spa water have been reported from greece.23 the early recognition of infection due to legionella plays a major role in its treatment and preventing mortality in patients with any underlying disease.24,25 in this case , the pcr method was lifesaving for the patient because it confirmed the infection , leading to the administration of the right treatment , whereas the first urine antigen test was misleading . this way of diagnosing legionella has been well established in previous published studies , and it should be applied in such cases where a differential diagnostic problem exists.26,27 legionnaires disease is an acute bacterial infection generally caused by l. pneumophila , primarily involving the lower respiratory tract . outbreaks have been described related to a common source of contamination.1 erythromycin has been the treatment of choice ever since a retrospective study of the original outbreak in philadelphia indicated a lower mortality rate with this antibiotic . because legionella is an intracellular pathogen , antibiotics that penetrate intracellularly are likely to be active against this pathogen . both fluoroquinolones and macrolides penetrate cells well , and fluoroquimolones achieve high intracellular levels and have a lower minimum inhibitory concentration against legionella than erythromycin.28 they are more active than erythromycin in inhibiting l. pneumophila in different intracellular models . three observational studies with a total of 458 patients have indicated that fluoroquinolones ( mainly levofloxacin ) are associated with a superior clinical response when compared with that of macrolides ( erythromycin and clarithromycin ) , as evidenced by a shorter time to apyrexia , shorter hospital stay , and fewer drug - related complications.2931 however , no randomized trials have been performed yet . although it has been traditional to add rifampin to erythromycin to treat severe legionellosis , an observational cohort study revealed that the addition of rifampin to clarithromycin was associated with more side effects and a longer hospitalization stay than erythromycin alone.30,32 in all cases of severe , life - threatening pneumonia , prompt administration of appropriate antibiotics is associated with improved outcomes . this case should urge every clinical doctor to study carefully the medical history of a patient and consider alternative diagnosis when the patient is not responding to the initial treatment . flouoroquinolones have established an equal if not superior therapeutic profile for legionellosis based upon published studies.2830 it should be mentioned at this point that a very important limitation of the study was that we were unable to take water samples from the spa in order to have a solid confirmation of the source of legionellosis . pcr is a useful tool in the hands of the clinical doctor and should be used where possible in suspicious cases , such as the above . also , early antibiotic treatment prevents the progression of the infection and should be administered upon admission . finally , flouoroquinolones , and in this case levofloxacin , should be considered an effective and efficacious treatment for legionellosis . written informed consent was obtained from the patient s next - of - kin for publication of this case report and any accompanying images .

the main purpose of this article is to provide a guide to theorems on global properties of solutions to the einstein - vlasov system . this system couples einstein s equations to a kinetic matter model . kinetic theory has been an important field of research during several decades in which the main focus has been on non - relativistic and special relativistic physics , i.e. , to model the dynamics of neutral gases , plasmas , and newtonian self - gravitating systems . in 1990 , rendall and rein initiated a mathematical study of the einstein - vlasov system . since then many theorems on global properties of solutions to this system have been established . this paper gives introductions to kinetic theory in non - curved spacetimes and then the einstein - vlasov system is introduced . we believe that a good understanding of kinetic theory in non - curved spacetimes is fundamental to a good comprehension of kinetic theory in general relativity . introductionthere are at least 40 types of legionella bacteria , half of which are capable of producing disease in humans . the legionella pneumophila bacterium , the root cause of legionnaires disease , causes 90% of legionellosis cases.case presentationwe describe the case of a 60-year - old woman with a history of diabetes mellitus and arterial hypertension who was admitted to our hospital with fever and symptoms of respiratory infection , diarrhea , and acute renal failure . we used real - time polymerase chain reaction ( pcr ) to detect l. pneumophila dna in peripheral blood and serum samples and urine antigen from a patient with pneumonia . legionella dna was detected in all two sample species when first collected.conclusionsince legionella is a cause of 2% to 15% of all community - acquired pneumonias that require hospitalization , legionellosis should be taken into account in an atypical pulmonary infection and not be forgotten . moreover , real - time pcr should be considered a useful diagnostic method .

bat guano , an excrement of the cave - dwelling bats forms the basis of the ecology inside the cave by acting as a food source for detritivorous microbes . it contains high content of organic carbon , nitrogen , phosphate , and potassium , . bacteria present in bat guano were reported to be involved in nitrification process and were also known as potential chitinase producer . a clone library based study in bat guano samples has revealed the presence of group 1.1a and 1.1b crenarchaeota , an efficient ammonia oxidizer , . analyzing bat guano is also important since they often harbor various pathogens which can be thread for speleologists , and tourists . although the microbial communities in diverse cave ecosystems have been studied , little is known about the microbial communities of bat guano heaps , and there has been no studies using high throughput sequencing technology . meghalaya is known to possess the largest and most diverse karst caves in the world . pnahkyndeng cave located in ri - bhoi district of meghalaya , india is a home of various bats and offering an ideal environment for studying the bat guano microbiota without any anthropological influence . samples were collected on february 2014 from the bat guano of pnahkyndeng cave ( 255722.70n , 915543.10e ) , nongpoh , ri - bhoi district , india . ten composite guano samples were collected from different places of the cave floor and the soil community dna was extracted separately using the fast dna spin kit for soils ( mp biomedical , solon , oh , usa ) . the freshly extracted dna was purified twice using 0.5% low melting point agarose gel and mixed to prepare a composite sample . final dna concentrations were quantified by the using a microplate reader ( bmg labtech , jena , germany ) . the v4 region of the 16s rrna gene was amplified using f515/r806 primer combination ( 5-gtgccagcmgccgcggtaa-3 ; 5-ggactachvgggtwtctaat-3 ) . amplicon was extracted from 2% agarose gels and purified using the qia quick gel extraction kit ( qiagen , valencia , ca , usa ) according to the manufacturer 's instructions . quality filtering on raw sequences was performed according to base quality score distributions , average base content per read and gc distribution in the reads . singletons , the unique otu that did not cluster with other sequences , were removed as it might be a result of sequencing errors and can result in spurious otus . chimeras were also removed using uchime and pre - processed consensus v4 sequences were grouped into operational taxonomic units ( otus ) using the clustering program uclust at a similarity threshold of 0.97 , . all the pre - processed reads were used to identify the otus using qiime program for constructing a representative sequence for each otus . the representative sequence was finally aligned to the greengenes core set reference databases using pynast program , . , 403,529 reads were classified at the phylum , 282,350 at the order , 188,406 at the family and 2926 sequences were classified at the species levels . classified otus belonged to 18 different phyla dominated by chloroflexi , crenarchaeota , actinobacteria , bacteroidetes , proteobacteria , and planctomycetes ( fig . 1 ) . analysis of bacterial communities revealed the two most dominant bacteria 's chloroflexi ( 29.97% ) and actinobacteria ( 22.55% ) , which are known to be a common inhabitant of cave microflora . other identified phyla include crenarchaeota ( 16.96% ) , planctomycetes ( 12.41% ) and proteobacteria ( 12.03% ) . chloroflexi was divided into 11 classes thermomicrobia , planctomycetia , gitt - gs-136 , ktedonobacteria , anaerolineae , tk10 , tk17 , s085 , chloroflexi , ellin6529 , and gitt - gs-136 . the most dominant otu within this phyla was denovo 317 , classified under the class thermomicrobia ( 40.52% ) followed by denovo 710 under the thermomicrobia ( 7.61% ) , denovo 235 under the genus thermogemmatisporaceae ( 7.18% ) and denovo 3 under the genus gemmataceae ( 2.98% ) . the dominant otu within this phyla was denovo 74 under the genus mycobacterium ( 29.39% ) followed by denovo 993 ( 8.27% ) and denovo 372 ( 5.27% ) classified under the genus acidimicrobiales and actinomycetales , respectively . only five otus were classified up to the species level ( mycobacterium llatzerense and mycobacterium celatum ) . a third dominant phylum in this sample was identified as planctomycetes comprising of 91 otus and 46,063 reads . seventeen archeal otus were classified under the order nrp - j , methanomicrobiales and methanosarcinales . four of them were identified at the genus level ( methanosarcina , haloquadratum , methanosaeta and methanocorpusculum ) . the phylogenetic tree based on the genus level relationships is provided as supplementary fig . 1 . previous study on archaeal communities present in bat guano identified many ammonia oxidizing bacteria but it was limited with a few number of clones . our analysis provides in - depth and high throughput identification of the bacterial communities present in bat guano . in the present study , we identified 18 bacterial phyla and most of the bacterial genus identified was known to be involved in nitrogen cycling as seen in previous study . a significant portion of otus still remains unclassified which indicates the possibility for the presence of novel species in pnahkyndeng cave . further studies like whole metagenome sequencing or functional metagenomics can illustrate the detailed information of this bacterial community . differential lipid composition between the apical and basolateral membrane domains of epithelial cell plasma membranes made it clear that membrane lipids are not laterally distributed in a homogeneous fashion . the lipid raft hypothesis was developed to explain lateral separation of bilayer lipids , and this idea quickly found applications in viral budding , endocytosis , and signal transduction ( reviewed in ) . in model membranes , lipids can separate into microscopically resolvable raft - like domains . plasma membrane surrogates formed by chemical membrane blebbing or cell swelling procedures also show phase behaviour [ 5 - 8 ] . similar domains are not evident upon direct observation of unperturbed plasma membranes in living cells , but the non - equilibrium nature of cell membranes , including endocytosis , exocytosis , and other motile processes , may prevent overt phase separation . likewise , quantitative analysis of lipid - anchored protein and lipid diffusion in cell membranes by fluorescence recovery after photobleaching ( frap ) , frster resonance energy transfer ( fret ) , and fluorescence correlation spectroscopy ( fcs ) [ 9 - 11 ] indicated that rafts in the plasma membrane of resting cells must be very small or ephemeral ( or both ) , forcing an evolution of the lipid raft hypothesis . these tiny clusters do not represent lipid phase separations but are probably short - range ordering imposed upon lipids by transmembrane proteins and cortical actin structures . thus , the current challenge for the field is to understand the interplay between protein and lipid that converts the exceedingly small , unstable clusters of components into larger , more stable membrane microdomains required for function . the recent development of sensitive quantitative microscopy methods has advanced our knowledge of lipid dynamics in resting cells . the diffusion of raft lipids ( e.g. , sphingomyelin ) and non - raft lipids ( e.g. , phosphatidylethanolamine ) was measured by an elegant fcs technique within regions as small as 30 nm in diameter using stimulation emission depletion fluorescence microscopy . the results indicate that raft lipids , but not non - raft lipids , are indeed preferentially trapped , albeit for short distances ( < 20 nm ) and for short periods ( 10 - 20 ms ) . homofret measurements , combining frap , emission anisotropy , and theoretical model fitting to test models of lateral organization in the membrane , were used determine the degree of clustering of glycosylphosphatidylinositol ( gpi)-anchored proteins in the plasma membrane . the formation of gpi - anchored protein nanoclusters ( of ~4 molecules or even less ) is an active process involving both actin and myosin , and these nanoclusters are nonrandomly distributed into larger domains of < 450 nm . additionally , high - speed single - particle tracking ( 50 khz ) revealed that gpi - anchored proteins , along with other membrane proteins , undergo rapid hop diffusion between 40 nm actin - regulated compartments , with a compartment dwell time of 1 - 3 ms on average . however , when gpi - anchored proteins were deliberately cross - linked by gold or quantum dot particles , they underwent transient confinement or stall ( stimulation - induced temporary arrest of lateral diffusion ) from a cholesterol - dependent nanodomain in a src family kinase mediated manner [ 17 - 19 ] . a recent study identified a transmembrane protein ( carboxyl - terminal src kinase [ csk]-binding protein ) involved in the linkage between the particle - cross - linked gpi - anchored protein , thy1 , and the cytoskeleton ( figure 1 ) . thy-1 crosslinking by streptavidin - coated quantum dots aggregates gpi lipid tails in the outer leaflet of the plasma membrane in a cholesterol - dependent manner . carboxyl - terminal src kinase ( csk)-binding protein ( cbp ) , a transmembrane protein , is recruited to or captured by thy-1 clusters along with src - family kinase substrates ( ks ) . cbp or ks ( or both ) are phosphorylated by src - family kinases ( sfk ) , enabling cbp to bind to actin filaments via an ebp50-erm ( ezrin / radixin / moesin - binding phosphoprotein 50-ezrin / radixin / moesin ) adaptor linkage resulting in a transient anchorage . when either cbp or the adaptors are dephosphorylated by an unspecified protein tyrosine phosphatase ( ptp ) the anchorage is terminated . the lipid envelope of influenza and hiv virions , but not those of the vesicular stomatitis virus ( vsv ) or semliki forest virus ( sfv ) , is enriched in raft - like lipids , leading to the notion that these viruses bud from lipid microdomains in the plasma membrane [ 21 - 25 ] . by contrast , the lipidomes of vsv and sfv are very similar to each other and to that of the plasma membrane suggesting that these viruses do not select or generate lipid raft domains for budding . the protein and lipid environment of the budding domains of hemagglutinin ( ha ) and hiv has been the source of several recent papers examining the process of viral budding using quantitative live - cell imaging techniques . influenza buds from ha clusters ( ranging up to micrometers in diameter ) regulated by ha transmembrane region length and palmitoylation . recent fluorescence lifetime imaging microscopy ( flim)-fret experiments in living cells indicate that ha colocalizes with lipid microdomain markers , further supporting the role of lipid - protein interactions in influenza virus budding . proton magic angle spinning nuclear magnetic resonance was used to detect a minor fraction ( ~10 - 15% ) of liquid - ordered membrane phospholipids in ha virions and virion lipid extracts at 37c . while lipid ordering increased at lower temperatures , it was not required for virion fusion with target membranes . progressive recruitment of cytoplasmic hiv-1 gag to the membrane , via post - translational acyl lipid modification and pip2/basic residue interactions , forms membrane domains that culminate in virion budding [ 31 - 33 ] . while the hiv-1 lipid envelope composition indicates enrichment in lipids and proteins associated with rafts , paradoxically , one report has failed to observe an enrichment of enhanced green fluorescent protein ( egfp)-gpi at gag domains in living cells , suggesting that the local lipid microenvironment may not exactly parallel the classic raft lipid composition . recent work has implicated the tetraspanin family of proteins in gag domain formation and function . tetraspanins , a widely expressed and highly conserved class of transmembrane proteins ( reviewed in ) , form tetraspanin - enriched microdomains ( tems ) through lateral tetraspanin - tetraspanin interactions and binding to non - tetraspanin membrane proteins . tetraspanins can be palmitoylated and the lipid environment within tems contains cholesterol , but gpi - anchored proteins and caveolin are not enriched in tems ( reviewed in ) . recently , cholesterol and tetraspanin palmitoylation were implicated in the confined diffusion and co - diffusion ( of two tetraspanin molecules ) of the tetraspanin cd9 . tetraspanins appear to induce order in the plasma membrane by virtue of protein clustering , but they likely also stabilize lipid microenvironments in the plasma membrane allowing for lateral organization of hiv-1 gag and virion budding . some lectin - based membrane domains form in the absence of post - translational lipid modifications or known lipid binding activity . dendritic cell - specific intracellular adhesion molecule-3-grabbing non - integrin ( dc - sign ) , a tetrameric c - type lectin with affinity for high - mannose glycans , forms microdomains on the plasma membrane [ 40 - 42 ] that serve as high - avidity binding sites for numerous pathogens . a previous report suggested that dc - sign interacts with lipid rafts , but this was based on detergent insolubility and cholera toxin colocalization assays , which generally do not faithfully report on intrinsic membrane lateral heterogeneity . also , dc - sign domains do not depend on cholesterol ( unpublished data ) . this result implies that dc - sign within domains does not exchange with the surrounding membrane . the source of this stability remains a mystery , and its cause may not reside in the membrane - apposed cytoskeleton but in extracellular cross - linking factors such as galectins ( reviewed in ) . dc - sign membrane domains that are multiplexed with another c - type lectin , cd206 ( unpublished data ) , appear to mediate the formation of fungipods , novel cellular protrusive structures involved in fungal recognition by dendritic cells ( figure 2 ) . thus , the lateral heterogeneity in membranes provided by rafts and other microdomains continues to provide surprising functional consequences . c - type lectins ( clrs ) form a type of plasma membrane domain that is not dependent on cholesterol . ( a ) plasma membrane domains containing mixtures ( yellow ) of dendritic cell - specific intracellular adhesion molecule-3-grabbing non - integrin ( dc - sign ) ( green ) and cd206 ( red ) are observed on a monocyte - derived dendritic cell ( dc ) by immunofluorescence . dc - sign domains are known sites of binding and entry for a range of pathogens including hiv-1 . ( b ) yeast cell wall material is sensed by these clr membrane domains , triggering a unique protrusive response , the fungipod . the image shows an example of a dc fungipod formed via cd206 ligation by a fixed saccharomyces cerevisiae particle ( zymosan ) , visualized by scanning electron microscopy ( 9500 ) . a variety of membrane domain forming systems have a wide gamut of lipid and protein constituents and possess a correspondingly broad range of functions . recent advances have shown that preferential lipid trapping or confinement in the resting plasma membrane occurs only on very small spatiotemporal scales . critical attention must be paid when determining if and when such confinement becomes biologically meaningful for processes such as endocytosis and signal transduction . while lipid ordering can be stabilized by oligomerization of membrane - associated proteins ( i.e. , gm1 crosslinking , influenza ha clustering ) , the lipids in these domains may still exchange between domain and surrounding membranes , making even these stabilized raft - like domains dynamic environments . at what point does a membrane domain become stable enough to be biologically relevant ? what is the range of protein and lipid turnover rates seen in membrane domains and are there different turnover rates for each constituent ? it is likely that a spectrum of membrane microdomains exists with different compositions and physical characteristics suited to their diverse purposes . the lipid species and their ordering within raft - like complexes appear to be key factors in determining intradomain cohesiveness and resultant domain size and lifetime .

v4 hypervariable region of 16s rdna was analyzed for identifying the bacterial communities present in bat guano from the unexplored cave pnahkyndeng , meghalaya , northeast india . metagenome comprised of 585,434 raw illumina sequences with a 59.59% g+c content . a total of 416,490 preprocessed reads were clustered into 1282 otus ( operational taxonomical units ) comprising of 18 bacterial phyla . the taxonomic profile showed that the guano bacterial community is dominated by chloroflexi , actinobacteria and crenarchaeota which account for 70.73% of all sequence reads and 43.83% of all otus . metagenome sequence data are available at ncbi under the accession no . srp051094 . this study is the first to characterize bat guano bacterial community using next - generation sequencing approach . evidence in support of the classical lipid raft hypothesis has remained elusive . data suggests that transmembrane proteins and the actin - containing cortical cytoskeleton can organize lipids into short - lived nanoscale assemblies that can be assembled into larger domains under certain conditions . this supports an evolving view in which interactions between lipids , cholesterol , and proteins create and maintain lateral heterogeneity in the cell membrane .

repetitive effortless vomiting is termed as rumination syndrome and is common in infants and mentally retarded individuals . it is associated with considerable morbidity in children who frequently miss the school , have history of multiple hospitalizations , and also suffer significant psychiatric morbidity . unfortunately , before reaching a correct diagnosis , a number of patients undergo unnecessary invasive testing and surgical treatments . symptoms are often confused with that of gastric motility disorders and diagnosis is often delayed due to poor awareness regarding adult rumination syndrome among physicians . here we are presenting a case that posed diagnostic confusion as already mentioned and showed improvement soon after the correct diagnosis was made . the main factors that helped in remission of symptoms were supportive psychotherapy and the firm attitude of the physician . a 26-year - old lady presented with her husband and mother with the complaints of recurrent and intractable vomiting since past 2 years . the vomiting was regular , occurring throughout the day , and it used to increase after meals . for the past 6 months , she was not able to hold even the liquid diet to the extent that few sips of water were sufficient to induce vomiting . along with vomiting , she also complained of episodic loss of consciousness , which she used to regain after 1 - 2 h. these episodes of unconsciousness specifically occurred when she was under some stress . besides this , she was not having any other complaint , e.g. , abdomen pain , fullness in the stomach after having meals , diarrhea or constipation , or swallowing difficulties . her history did not suggest any cardiac problem , focal neurological deficits , or convulsions . at the time of presentation , she was pregnant ( second trimester ) with normal fetal growth . her family history was unremarkable except that she had strained relations with her parents - in - law . though her husband was very supportive but for almost 1 year she was staying at her mother 's house due to this illness . since the onset of illness she underwent extensive laboratory investigations including upper gastro - intestinal endoscopy , barium meal , and mri brain but any of the tests did not reveal any finding that could explain the symptoms . results of her liver and kidney function tests were within normal limits since the onset of illness . in the past , she was treated with a number of drugs including antidepressants , antipsychotics , promethazine , proton - pump inhibitors , and prokinetics for optimal periods without any relief . her husband also took her for a vacation when suggested by a physician , but it also did not improve the situation . looking at such a long history , multiple consultations , and extensive laboratory work - up , she was interviewed in the presence of her husband . then it became clear that her vomiting was effortless ; in other words , she used to stand in front of a wash basin and without putting any finger or anything else in the mouth , used to expel all the food ( liquid or solid ) that she had . the vomiting act was not associated with lacrimation , salivation , cramping of the abdomen , cessation of respiration followed by hyperventilation , and the gag sound that is otherwise common during true vomiting . it always took her about half an hour to expel the food . to make the information clearer , she was made to drink a glassful of water and her vomiting was observed which confirmed the information provided by her husband . her general physical examination showed pale conjunctiva but we did not find any signs of induced vomiting . however , she was not meeting all criteria for the major depressive disorder according to dsm - iv tr . looking at the concern of her family members and her pregnancy , she was hospitalized and reassured . when she did not improve even after 2 days , in view of her pregnancy , oral feeding from a naso - gastric tube was started . to our surprise , she improved within few hours and started taking liquids orally without vomiting them . she also insisted on the removal of the naso - gastric tube , but due to our concerns for the maintenance of feeding , it was kept for 2 days . she kept on taking liquid diet orally as well as through the tube and the symptoms did not recur . now her tube was removed and she was insisted to have solid food . for the next 2 days , she even had solid food without vomiting anything ; at this juncture , supportive psychotherapy was started . she was discharged after 3 days with the advice to attend regular supportive psychotherapy sessions . the diagnosis often poses a challenge to the physicians , especially when encountered in adults with normal intelligence and is often confused with gastroparesis and gastroesophageal reflux . in these adults , it is commonly associated with psychiatric morbidity in themselves or in their family members . one common psychiatric problem is personality disturbances with high scores on hypochondriasis and depression sub - scales . previous literature suggests that these patients often show multiple physician consultations and diagnosis is often delayed by years , and the food is usually expelled within few minutes of having it . could find manometric abnormality only in one - third of patients while levine et al . the present case , gastroscopy , barium meal and mr scan of head were done before presenting to us . however , none of them showed any abnormality . in view of the available literature and her pregnancy the treatment of adult rumination syndrome consists of reassurance , behavior therapy , psychotherapy , and relaxation therapies . these may improve a number of patients while others suffer persistence of symptoms.[36 ] nissen fundoplication has been reported to provide complete relief in the symptoms in one report . this patient was also shown to a number of physicians and was given all kinds of medical treatment including gastrokinetics , antacids , antidepressants , and benzodiazepines without any improvement . the relief was obtained with reassurance , supportive psychotherapy , and our firm attitude to take care of the patient 's health . in conclusion , adult rumination syndrome may be more prevalent than it appears and adequate knowledge of this syndrome among the physicians is necessary to reach to a diagnosis and early intervention . in addition , further studies are required to assess therapeutic benefits of various modalities in this entity . an atlanto - axial synovial cyst is very rare . since the first report of this lesion by onofrio and mih1 ) in 1988 , to our knowledge . however , the current report described a patient with a large hemorrhagic cystic mass which was seen around prevertebral space of the atlantoaxial joint on the left side and the obstruction of the nasopharyngeal cavity on cervical magnetic resonance image ( mri ) . we report a symptomatic case associated with rheumatoid atlanto - axial subluxation , which regressed after the surgical management . a 72-year - old woman presented with sudden severe headache in her left occipital area with dyspnea . a laboratory examination of the patient 's blood revealed no remarkable abnormality indicative of inflammation , but high - titer of rheumatoid factor ( 95.9 iu / ml ) . seven months ago before visiting to our department , she checked brain mri due to sudden attack of severe headache on left occipital area , which revealed non - specific lesion except rheumatoid pannus with small prevertebral cyst of c1 - 2 junction ( fig . , there was a large hemorrhagic cystic mass around prevertebral space of the atlanto - axial joint on the left side , obstructing the nasopharyngeal cavity on cervical mri ( fig . the cystic mass was connected to atlanto - axial joint capsule on axial view of computed tomography ( ct ) . multiple bony erosion , rheumatoid atlanto - axial instability including left tilted c1 - 2 subluxation and cranial settling were demonstrated . and there were loss of lordosis and retrolisthesis in c 3 - 4 , 4 - 5 ( fig . 3 ) . in the first operation stage , considering patient 's dyspnea , aspiration of the cystic lesion was performed via transoral approach with otolaryngology surgeon ( fig . after 0.5 cm - sized mucosal incision on left side oropharyngeal wall , residual material of the cyst was squeezed by forceps and removed by suction tools . in the second operation stage , there were rheumatoid atlanto - axial instability and retrolisthesis in c 3 - 4 , 4 - 5 , so it was followed by posterior occipito - cervical fusion that connected from occiput to c5 , using vertex screw & rod system and iliac bone graft ( fig . 5 ) . the patient was tolerable on her postoperative course and showed good respiration and relieved headache . cervical synovial cysts are rare and located at the c1 - 2 junction or lower cervical spine . only 24 cases of synovial cysts of the c1 - 2 junction have been reported in the literature5 ) . moreover , the pathogenesis of spinal synovial cysts remains unclear but is thought to be attributable to degenerative changes of the facet joints or excessive joint motion2,3,5,7 ) . it revealed multiple bony erosions and a large pannus formation of c1 - 2 junction on this case . this patient also showed atlanto - axial instability caused by rheumatoid arthritis , such as left tilted antlanto - axial subluxation and basilar impression . the atlantoaxial articulation is a true synovial joint and is responsible for a large proportion of normal cervical mobility . the etiology of articular cysts is unclear , but they are assumed to be degenerative because minor chronic damage to articular surfaces produces a reactive proliferation of synovium or fibrocartilage that includes loculated collections of mucinous fluid . so , we hypothesized that hemorrhagic event was developed as a result of microtrauma caused by rheumatoid atlantoaxial instability . it is known that the higher titer of rheumatoid factor , the more destructive manifestations of joint occur1 ) . the patient was in the controlled state in serologic inflammatory marker , such as esr(erythrocyte sedimentation rate ) and crp ( c - reactive protein ) . however , the high titer of rheumatoid factor , in spite of long standing medication of rheumatology , was related factor of progressive destruction of joints . we report a rare case of large hemorrhagic cyst on prevertebral space of left side c1 - 2 area associated with rheumatoid arthritis causing airway obstruction and left occipital pain , successfully managed anterior and posterior approach . we suggest repeated microtrauma due to atlanto - axial subluxation associated with rheumatoid arthritis as a main cause of hemorrhagic event on the cyst .

rumination syndrome is known to exist in infants and mentally retarded adults since long time . in past few years , some reports appeared that showed its existence in adult patients also . it is frequently confused with the intractable vomiting in adults and misdiagnosis leads to delay in appropriate management . we are here describing the case of a female patient with rumination syndrome where specific points in the history delineated the presence of this illness and helped in appropriate management . the patient became symptom free soon after the diagnosis was reached . synovial cyst on prevertebral space of c1 - 2 joint is rare but may be associated hemorrhagic event . we describe a case of a 72-year - old woman who presented with sudden severe headache in her left occipital area with dyspnea . she had rheumatoid arthritis for 14-years . large hemorrhagic cystic mass was seen around prevertebral space of the atlantoaxial joint on the left side on cervical mri ( magnetic resonance image ) and it obstructed the nasopharyngeal cavity . aspiration of the cystic lesion was performed via transoral approach , followed by posterior occipito - cervical fusion . the specimen was xanthochromic , suggesting old hemorrhage . the patient was tolerable on her postoperative course and showed good respiration and relieved headache . we suggest that repeated microtrauma due to atalantoaxial subluxation associated with rheumatoid arthritis as a main cause of hemorrhagic event on the cyst .

methanol has the potential to be an efficient fuel for direct methanol fuel cells ( dmfcs ) with many applications ranging from small portable devices to large stationary power plants . however , one major challenge that dmfc is facing is the fragility of the proton exchange membrane which is very thin in a dmfc . the thickness is typically less than 100 m . a tiny crack or defect could fail the whole fuel cell . ionic liquids ( ils ) can be a replacement of the conventional proton exchange membrane to overcome the risk of fragility . used as the electrolyte , ionic liquids possess many advantages such as high ionic conductivity , chemical stability , and resistance to high temperature . the current generation ionic liquids are stable even in the environment of moisture and air , making them ideal for general use as electrochemical media [ 3 , 4 ] . in developing il - based dmfc , the concentration of methanol must be controlled and monitored at several locations : the fuel feed , the electrolyte , the electrode assembly , and so forth . it is critical to develop a fast sensing technique for methanol in the environment of ionic liquids . in this paper the technique is based on potential step analysis and is reliable even when water is present in the ionic liquids . chemicals and electrodes were purchased directly from the manufacturers or from venders such as fisher scientific and vwr : 1-butyl-3-methylimidazolium tetrafluoroborate ( basf quality , 98% ) from aldrich , methanol ( reagent grade acs ) from pharmco - aaper , phosphate buffer saline ( 25x ) from thermo scientific , l(+)-ascorbic acid ( 99% ) from acros , potassium tetrachloroplatinate ( ii ) 98% from aldrich , sulfuric acid 2.0 n from labchem , and gold disk electrode ( 2 mm dia . ) from ch instruments . 1-butyl-3-methylimidazolium tetrafluoroborate ( bmimbf4 ) is one of a few ionic liquids with relatively small viscosity , but it is still very viscous when compared to aqueous solutions . the presence of trace amount of less viscous methanol may reduce the viscosity or increase the conductivity significantly . therefore , our most straightforward thinking was to sense methanol by the change in conductivity . our thought was confirmed as the conductivity of bmimbf4 showed a linear relationship with the concentration of methanol in it . as shown in figure 1 , the conductivity would be a good indicator of methanol content if there was no interference of water . secondly , water is produced on the cathode and it could cross over to the electrolyte and to the anode ( 2 ) . thirdly , ionic liquids including bmimbf4 absorb water from the environment and the water content in bmimbf4 could reach as high as 0.29 wt% : ( 1)anode : ch3oh+h2oco2 + 6h++6e ( 2)cathode : 32o2 + 6h++6e3h2o water will also affect the conductivity of ionic liquids as reported before . for this reason , the conductivity sensing method has little use in a dmfc . a practical method must be water - resistant . we found a potential step method with pt - nanoparticle - coated au - nanoporous film ( pgnf ) was a reliable sensing method for methanol even when water was present . methanol was oxidized on pgnf electrode when the potential step was applied ( 1.9 v versus ag / agcl with saturated kcl solution ) . the current became smooth after 1 second and it was still measurable with amplitude of 175 a after 2 seconds . our previous study has found that the adsorption / desorption and double layer charging were dominant within the first 2 seconds of potential step . in the following report , the potential was fixed at 1.9 v versus ag / agcl ( saturated kcl ) and the sampling time at 2 seconds . under the small a / v ( electrode area to electrolyte volume ) condition and with a planar electrode , the current in a single potential step chronoamperometry can be predicted according to the cottrell equation : ( 3)it = nfad01/2c01/2t1/2 . in ( 3 ) , n is the number of electrons involved in redox reaction , f is the faraday constant , a is the electrode area , d 0 is the diffusion constant , c 0 is the bulk concentration , and t is the sampling time . if t is fixed in the potential step analysis , the sampling current i(t ) should be directly proportional to the concentration c 0 or the concentration of methanol in bmimbf4 . the current at the sampling time ( 2 seconds ) at various concentrations of methanol in bmimbf4 is shown in figure 3 . the sampling current did not change too much when the methanol concentration was smaller than 1 m. desorption / adsorption and double layer charging were predominant at the sampling time when the concentration was low . when the concentration was above 1 m , the electrooxidation of methanol became the predominant process and the sampling current had a linear relationship with the methanol concentration . the potential step method described above not only had a good linearity but also was waterproof . when water was added to the methanol solution in bmimbf4 , it did not interfere with the oxidation of methanol . the only effect of water was that it changed the molarity of methanol in bmimbf4 . as shown in figure 4 , the linearity was not destroyed by the presence of water , even when the water content was as high as 2.4 m. conductivity monitoring was a reliable method in sensing methanol in ionic liquids , but its use was limited to the situation without the presence of water . a potential step method with properly selected potential step and sampling time could be a more practical method to analyze methanol concentration in ionic liquids especially when the methanol concentration was high so that the sampling current was predominantly contributed by the methanol oxidation . epithelial monolayer ( em ) is the largest body tissue lining many organs in the human body . in the intestine , em provides protection to the internal body from toxic and infectious agents while at the same time it facilitates absorption of digested food and water from the gut . epithelial monolayer integrity and paracellular transport are the important features that can be protected and maintained with the help of epithelial barrier function . epithelial cells are connected with each other by four types of junctions , that is , desmosomes , gap junctions , adherens junctions , and tjs [ 24 ] . tight junctions are impermeable and control the movement of molecules and ions via a paracellular pathway . until recently , tight junction functions were categorized as fence as they separate the apical and basolateral cell surface domain defining cell polarity or a barrier due to their control over solutes and liquid flow through the paracellular space between the epithelial cells [ 58 ] . however tjs are not restricted to the fence and barrier function but have been defined to participate in signal transduction processes , gene expression , cell proliferation , and differentiation [ 911 ] . various unidentified external and internal regulators impair the normal function of tjs causing loss of water and solute in the passive manner that leads to leaky - flux watery diarrhea . tight junction ( tj ) is a complex structure constituting of growing numbers of components , including integral membrane proteins ( claudins , occludin , and junctional adhesion molecules jams ) and peripheral membrane proteins . the peripheral membrane proteins include ( 1 ) scaffold pdz ( postsynaptic density protein ( psd95 ) , drosophila discs large tumor suppressor ( dlg1 ) , and zonula occludens-1 protein ( zo-1 ) ) , multi - pdz domain protein-1 ( mupp-1 ) , and membrane - associated guanylate kinase ( magi-1 ) ; ( 2 ) no - pdz expressing proteins such as cingulin , symplekin , atypical protein kinase c , ras - related protein rab-3b ( rab-3b ) , ras - related protein rab-13 ( rab-13 ) , phosphatase and tensin homolog ( pten ) , and 7h6 antigen ; ( 3 ) cell polarity molecules asip / par-3 , partitioning defective 6 homolog alpha ( par-6 ) , and pals1-associated tj protein ( patj ) [ 13 , 14 ] . besides these proteins , tricellulin protein has recently been identified at the epithelial cell junctions with involvement in the barrier function . claudin family so far includes 24 reported members in different types of mammalian cells ; among them 21 are known components of tj in em in the kidneys , liver , brain , and intestine . these are involved in various physiological processes such as regulation of paracellular permeability and conductance . claudins are found in homo and heterotypic manner in single tj [ 13 , 26 ] . they can be divided into two main categories , pore - sealing and pore - forming claudins . claudin-1 , -3 , -4 , -5 , -7 , and -19 are known as pore - sealing claudins and an increased expression of these claudin proteins leads to increased tightness of em and increased transepithelial electrical resistance ( teer ) and decreases solute permeability across the monolayer [ 2731 ] . on the other hand , claudin-2 and -15 are considered as the pore - forming claudins , because of their ability to form paracellular anion / cation pores as well as water channels and therefore they decrease epithelial tightness and increase solute permeability [ 13 , 32 ] . epithelial barrier dysfunctions occur in inflammatory bowel diseases ( ibds ) like crohn 's disease ( cd ) or ulcerative colitis ( uc ) that contribute to leaky - flux diarrhea , that is , loss of solutes and water in increased amount dependent upon the components of tj proteins . downregulation of pore - sealing claudins ( e.g. , 4 , 5 , and 8) while upregulation of pore - forming claudin-2 is observed in active crohn 's disease patients [ 33 , 34 ] . numerous studies have reported leaky diarrhea in patients undergoing immunosuppressive therapy after organ transplantation [ 3538 ] . recently our group has reported mycophenolic acid- ( mpa- ) mediated increased expression of myosin light chain kinase ( mlck ) , myosin light chain-2 ( mlc-2 ) , and mlc-2 phosphorylation and redistribution of zo-1 and occludin in caco-2 and in hek-293 cells [ 39 , 40 ] as a possible mechanism of diarrhea in patients undergoing immunosuppressive therapy . transcription factors ( tfs ) play an important role in the gene regulation at the promoter level working either as an activator or as a repressor of a specific gene . the current review will focus major claudins family members ( table 1 ) and their regulators , which alter claudins gene activity at promoter level and therefore modulate tjs structure and function . claudin-1 protein is a key constituent of tjs and its altered expression is reported in a variety of cancers , most prominently colorectal cancers [ 13 , 17 , 41 , 42 ] . promoter region ( 1160 bps to 850 bps ) of claudin-1 consists of putative binding sites for caudal - related homeobox ( cdx-1 , -2 ) , gata4 , and t - cell factor / lymphoid enhancing factor-1 ( tcf / lef-1 ) transcription factors . there is a direct correlation between claudin-1 and cdx-2 expression in human colon cancer patient . cdx-2 is a homeobox domain - containing nuclear transcription factor that plays an important role in intestinal development by regulating the proliferation and differentiation of intestinal cells [ 4345 ] , and it is expressed in all cells along the crypt villus axis . cdx-2 transcriptional activity is controlled through mitogen - activated protein kinase / extra cellular signal regulated kinase pathway ( mapk / erk pathway ) which phosphorylates it at ser-60 position and resultantly reduces cdx-2 transcription activity in crypt and lowers villus cells . on the other side , cyclin - dependent kinase 2 ( cdk2 ) phosphorylates cdx-2 at ser-281 which coordinates cdx-2 polyubiquitination and degradation by the proteasome [ 43 , 4649 ] . specificity protein-1 ( sp-1 ) is the first identified transcription factor of specificity protein / krppel - like factor ( sp / xklf ) family , consisting of 785 amino acids ( aa ) with molecular weight of 100 to 110 kda . sp-1 's dna binding domain is the most conserved among other domains of sp family members which consisted of cy2his2 zinc ( zn ) fingers . mutational analysis has revealed that zn fingers 2 and 3 are essential for dna binding activity . sp-1 binds to the gc - rich elements that are common regulatory elements in the promoters of numerous genes . sp-1 binds its individual binding sites as a multimer and is capable of synergic activation of promoters containing multiple binding sites and regulates transcription by dynamically recruiting and forming complexes with many factors associated with transcription . normally sp-1 has been described as a transcriptional activator but it can also act as a repressor . claudin-1 promoter region ( 138 to 76 bp ) contains sp-1 binding site and a mutation in this region results in a significant loss of claudin-1 transcription . claudin-2 , also known as leaky protein , forms paracellular water channels in tjs and mediates paracellular transport of water molecules across the em . it is also involved in many signaling pathways , including vitamin d receptor , epidermal growth factor receptor ( egfr ) , and c - jun n - terminal kinases ( jnk ) signaling pathways , and contributes to inflammatory bowel disease and colon cancer [ 33 , 5558 ] . salmonella infection facilitates bacterial invasion across the em by inducing claudin-2 expression and altering its localization in tjs which is reversible by specific inhibitors ( egfr ( gefitinib ) and jnk ( sp600125 ) ) , making claudin-2 as a potential therapeutic target to prevent bacterial invasion and inflammation . interleukin-6 ( il-6 ) increases tj permeability of caco-2 monolayer from the basal side by inducing caludin-2 expression . il-6 activates the mitogen - activated protein kinases / extracellular signal - regulated kinases ( mek / erk ) pathway by inducing phosphorylation of erk and phosphatidylinositol 3-kinase ( pi3k / akt ) by phosphorylating akt , which in turn enhances cdx-2 expression . in the claudin-2 promoter region ( 1067 to 1 ) , four cdx-2 ( cdx - a , -b , -c , and -d ) , stat , and nuclear factor - kappa - light - chain - enhancer of activated b cells ( nf-b ) putative binding sites are identified . il-6 induced expression of claudin-2 can be reversed by using either specific inhibitors of mek / erk and pi3k / akt pathways ( u0126 ( a mek inhibitor ) and ly294002 ( a pi3k inhibitor ) ) or site directed mutagenesis in the putative cdx-2 binding sites in the promoter region of claudin-2 gene . a sp-1 binding site ( 112 and 74 bps ) in the promoter region of claudin-3 is crucial for its activation . claudin-3 expression is significantly decreased at mrna and protein levels , by knocking down the sp-1 with sirna , indicating an essential role of sp-1 in claudin-3 activation . claudin-4 is mainly expressed in the em of colon , renal tubules , mammary gland , and thyroid gland and is considerably raised in their cancers . there are two known sp-1 binding sites ( between 105 and 49 bps ) in the promoter region of claudin-4 . caludin-5 is mostly expressed in the tjs of em of pancreatic acinar cells , alveolar lung cells , colon , and endothelial cells forming the blood - brain barrier and endoneurial blood - nerve barrier . in colonic regions , its expression is mainly involved in the paracellular sealing of tjs [ 33 , 6163 ] . both downregulation and redistribution of claudin-5 can alter tjs structure leading to barrier dysfunction in active crohn 's disease . forkhead box ( foxo ) gene family members are potent transcriptional activators with four known members ; foxo1 ( also known as foxo1a ) , foxo3 ( also known as foxo3a ) , foxo4 , and foxo6 which bind to conserved consensus core recognition motif ttgtttac [ 6466 ] . four pairs of putative binding sites for foxo and tcf--catenin ( tcf--catenin act as a stabilizer ) are identified in the three regions of claudin-5 promoter ( region 1 , position 2,906/2,871 ; region 2 , position 2,317/2,287 ; region 3 , position 1,103/1,008 ) . both foxo1 and e74-like factor 3 ( elf3 ) belongs to e26 transformation - specific sequence ( ets ) family of transcription factors and binds to the ets binding site in the promoter region ( 150 bps ) of claudin-7 . members of ets family are mainly involved in cell differentiation , proliferation , and cell transformation . regulation of the target genes by ets factors depends upon their activation by mapk and their association with other cofactors [ 68 , 69 ] . an essential role of elf3 is reported in epithelial cell differentiation [ 7072 ] and small interference rna ( sirna ) treatment downregulates the claudin-7 expression validating the central role of elf3 in claudin-7 activation . claudin-15 is a pore - forming protein expressed in the em of intestine , liver , and kidney tissues . four putative binding sites ( bs1 - 4 ) of transcription factor hepatocyte nuclear factor 4 alpha ( hnf4 ) are present in the ( 693 to 47 bps ) region of claudin-15 promoter . hnf4 is considered as an important regulator of em barrier integrity and is involved in the regulation of metabolism , cell junction , differentiation , and proliferation of liver and intestine epithelial cells . both animal model and ibd patients ' biopsy studies have shown that an altered expression of hnf4 directly influences the expression and distribution of claudin-15 . 5060% of filtered mg , and 3035% of filtered ca are reabsorbed into the body by thick ascending limb , the loop of henle . claudin-16 and -19 play a main role in the regulation of mg reabsorption and loss of either claudin-16 or -19 leads to excessive renal waste of mg . four putative transcription factor ( not characterized , ap2 , nf - e , and sp-1 ) binding sites are located between 139 and 75 in the promoter region of mouse claudin-19 . however , only sp-1 is described for having an important role in the expression of claudin-19 and a mutation in sp-1 binding site significantly reduces the claudin-19 expression . tight junctions play an important role in the regulation of paracellular movement of molecules across the em , impart mechanical strength , maintain the polarity of cells , and prevent the passage of unwanted molecules and pathogens through the space between the plasma membranes of adjacent cells . the efficiency of the junction in preventing ion passage increases exponentially with the number of strands of claudins family proteins which are having important role in the structure as well as controlling paracellular movement across the tight junctions . altered expression of claudins family proteins in tjs plays a key role in numerous abnormalities like cancers , ibds , and leaky diarrhea and a better understanding of their regulatory mechanism could help in designing innovative therapeutic strategies .

the development of direct methanol fuel cells required the attention to the electrolyte . a good electrolyte should not only be ionic conductive but also be crossover resistant . ionic liquids could be a promising electrolyte for fuel cells . monitoring methanol was critical in several locations in a direct methanol fuel cell . conductivity could be used to monitor the methanol content in ionic liquids . the conductivity of 1-butyl-3-methylimidazolium tetrafluoroborate had a linear relationship with the methanol concentration . however , the conductivity was significantly affected by the moisture or water content in the ionic liquid . on the contrary , potential step could be used in sensing methanol in ionic liquids . this method was not affected by the water content . the sampling current at a properly selected sampling time was proportional to the concentration of methanol in 1-butyl-3-methylimidazolium tetrafluoroborate . the linearity still stood even when there was 2.4 m water present in the ionic liquid . human gastrointestinal tract is covered by a monolayer of specialized epithelial cells that constitute a protective barrier surface to external toxic and infectious agents along with metabolic and digestive functions . intercellular junctions , among epithelial cells , such as desmosomes , adherens , gap , and tight junctions ( tjs ) , not only provide mechanical integrity but also limit movement of molecules across the monolayer . tj is a complex structure composed of approximately 35 different proteins that interact with each other at the apical side of two adjacent epithelial cells . claudin family proteins are important members of tj with so far 24 known isoforms in different species . claudins are structural proteins of tj that help to control the paracellular movement by forming fence and barrier across the epithelial monolayer . altered function of claudins is implicated in different form of cancers , inflammatory bowel diseases ( ibds ) , and leaky diarrhea . based on their significant role in the molecular architecture of tj , diversity , and disease association , further understanding about claudin family proteins and their genetic / epigenetic regulators is indispensable .

drug induced liver injury ( dili ) is a major issue worldwide , both for patients and health providers . it is one of the primary causes for attrition during clinical and preclinical studies and the main reason for drug withdrawal from the market . dili is divided into types , ( i ) hepatocellular , ( ii ) cholestatic , or ( iii ) mixed ( hepatocellular and cholestatic ) , according to the type of liver damage and the clinical chemistry biomarker alterations . the cholestatic and mixed hepatocellular and cholestatic type are the two most severe manifestations of dili and yield almost half of the recorded cases of dili . cholestatic liver injury , or more simply cholestasis , is the disruption of the bile flow , which might be either due to biliary tract obstruction or to complications in bile acid uptake . while the mechanistic basis for hepatocellular dili is still a mystery for the majority of the cases , more knowledge exists for cholestatic dili . there is growing evidence for a vast amount of cholestasis cases pinpointing the important role of hepatic transporters . basolateral transporters are responsible for the uptake of drugs and other endobiotics and xenobiotics from the blood , influencing the exposure of the hepatocyte to potential damage . canalicular transporters regulate the hepatic clearance , as well as the secretion of bile salts and bile conjugates into bile . any disturbance of the transporters physiological function may result in the accumulation of potentially harmful bile products that can finally cause cholestasis.figure 1 provides an overview on the respective location of hepatocyte transporters . blue symbols represent mainly the canalicular transporters , and red symbols , the basolateral ones . mrp16 multidrug resistance - associated proteins 16 , ost/ost organic solute transporter , bsep bile salt export pump , bcrp breast cancer resistance protein , mate1 multidrug and toxin extrusion transporter 1 , abcg5/g8 atp - binding cassette subfamily g member 5/8 , mdr3 multidrug resistance protein 3 , p - gp p - glycoprotein , atp8b1 atpase - aminophospholipid transporter , oatp organic anion transporting polypeptide , ntcp sodium ( na ) taurocolate cotransporting polypeptide , oct organic cation transporter 1 , oat organic anion transporter . the most important one , due to its pivotal role in bile salts clearance , is the bile salt export pump ( bsep ) . apart from bsep , there is evidence for the implication of other canalicular efflux transporters such as the multidrug resistance - associated protein 2 ( mrp2 ) , breast cancer resistance protein ( bcrp ) , multidrug resistance protein 3 ( mdr3 ) , and p - glycoprotein ( p - gp ) . mdr3 functions as an atp - dependent phospholipid flippase , translocating phosphatidylcholine from the inner to the outer canalicular membrane . canalicular phospholipids are then solubilized by canalicular bile salts to form mixed micelles , protecting cholangiocytes from the detergent properties of bile salts . while p - gp is also not transporting bile salts , it is implicated in cholestasis because of its large amount of substrates and inhibitors which cause drug drug interactions that disrupt the smooth function of the hepatocyte . the basolateral transporters play also an important role , both the uptake transporters , such as organic anion transporting polypeptides 1b1 , 1b3 , and 2b1 ( oatp1b1 , 1b3 , and 2b1 ) and sodium ( na ) taurocolate cotransporter ( ntcp ) , and the efflux transporters , like multidrug resistance - associated protein 3 and 4 ( mrp3 and mrp4 ) . in particular , in cases of cholestasis , the basolateral uptake transporters ntcp and oatp1b1 have been found down - regulated . however , in this case , oatp1b3 is up - regulated as a compensatory mechanism for the elimination of xenobiotics from sinusoidal blood . on the contrary , in cases of cholestasis , mrp3 and mrp4 are up - regulated to facilitate the efflux of the toxic bile salts out of the hepatocyte . thus , simultaneous inhibition of several of these transporters could induce drug toxicity due to inadequate elimination from the blood or increase the cholestatic effect due to accumulation of bile salts in the hepatocyte . consequently , drug - induced liver injury and cholestasis are important toxicity alerts to be considered in drug development . interestingly , there are only a few computational studies for the prediction of cholestasis reported in literature . with respect to the involvement of hepatic transporter , there are some in vitro studies correlating cholestasis with transporter inhibition , such as bsep , mrp3 , mrp4 , and ntcp . also several in silico studies for the identification of potentially cholestatic compounds via modeling of transporters and then associating them with the cholestatic effect of their inhibitors have been conducted . a characteristic example is the study by greupink et al . in 2012 , who developed a pharmacophore approach for ntcp in order to identify potentially ntcp inhibitors . under the same principles , in 2014 ritschel and colleagues performed a 3d ligand - based pharmacophore model for bsep inhibition . however , in most of these cases the amount of validated drugs is small and what is basically described is the association between transporter inhibition and cholestasis . thus , as the respective is associated with cholestasis , it is assumed that an inhibitor is causing cholestasis . most recently , muller et al . , in order to model dili , also modeled some more hepatotoxicity end points , including cholestasis . moreover , mulliner et al . presented a multilevel modeling approach for dili , where cholestasis was also included as a morphological hepatobiliary finding . however , examining the liver transporters contribution was not within the scope of their work . dilisym ( www.dilisym.com ) is a mechanistic mathematical model of dili , that has been used to investigate the effects of bsep inhibition on drug - induced liver injury , as well as on bile acid - mediated dili . developed a multiscale , liver - centric in silico modeling framework for acetaminophen pharmacology and metabolism that can be extended in predicting hepatotoxicity due to acetaminophen overdosing . in this study we present a classification scheme in order to predict cholestasis from a public data set , using physicochemical descriptors as well as predicted transporter inhibition profiles . for the latter we used our in house classification models for bsep , bcrp , p - gp , oatp1b1 , and oatp1b3 . for compiling the dili training data set we searched in pubmed ( http://www.ncbi.nlm.nih.gov/pubmed ) , google , scopus ( https://www.scopus.com/ ) , and the sider database v2 using the search terms : drug - induced cholestasis or the retrieved publications were then investigated manually for human data , i.e. compounds that are positive or negative for drug - induced cholestasis in humans . those compounds in principle drugs were added to those obtained from the sider v2 database . unfortunately , cholestasis is an end point that is not widely examined in terms of experimental or in silico studies that would potentially guide us to big data sets . thus , even though we were able to compile several drugs positive for cholestasis , there was almost no information in terms of the negatives . on the other hand , dili in general is studied quite extensively and there are several respective data sets . since choleastasis is a possible manifestation for dili , we can consider safely that any compound negative for dili will definitely be also negative for cholestasis . thus , the negative compounds for dili that we had compiled and curated in a previous work were also used as negatives for this study . the data set was carefully curated according to the following rules : all inorganic compounds were removed based on their chemical formula in moe 2014.09.salt parts and compounds containing metals and/or rare or special atoms were removed and the chemical structures were standardized using the standardiser tool created by francis atkinson.duplicates and permanently charged compounds were removed using moe 2014.09 . here we have to note that stereoisomers , even if biologically can be considered as different compounds , were considered as duplicates , since they give the exactly same vector of descriptors . if two ( or more ) stereoisomers are of the same class , only one was kept . if they were of different classes , they were all removed.3d structures were generated using corina ( version 3.4 ) , and their energy was minimized with moe 2014.09 , using default settings , but changing the gradient to 0.05 rms kcal/(mol a ) . all inorganic compounds were removed based on their chemical formula in moe 2014.09 . salt parts and compounds containing metals and/or rare or special atoms were removed and the chemical structures were standardized using the standardiser tool created by francis atkinson . here we have to note that stereoisomers , even if biologically can be considered as different compounds , were considered as duplicates , since they give the exactly same vector of descriptors . if two ( or more ) stereoisomers are of the same class , only one was kept . 3d structures were generated using corina ( version 3.4 ) , and their energy was minimized with moe 2014.09 , using default settings , but changing the gradient to 0.05 rms kcal/(mol a ) . in addition , the existing chirality was preserved . after these curation steps 152 compounds remained as positives for cholestasis . the negatives for dili , and subsequently for cholestasis , were 466 compounds . however , when uniting the data , there were compounds with contradictory class assignments . these compounds were removed from the data set , yielding a data set of in total 578 compounds ( 131 positives and 447 negatives ) . the compiled data set is provided in the supporting information . recently and after having already compiled our training set for cholestasis and developed the respective model a data set covering multiple levels of hepatotoxicity was published by mulliner and co - workers . the data are hierarchically clustered by the authors into three levels of hepatotoxicity : level 0 corresponds to general hepatotoxicity , level 1 corresponds to clinical chemistry findings and morphological finding as distinguished parts of general hepatotoxicity , and level 2 discriminates both clinical chemistry and morphological findings into hepatocellular and hepatobiliary injury . we use only clinical data , i.e. the human data , of morphological findings for hepatobiliary injury as an external test set for validating the developed cholestasis model . once more , we performed chemical data curation and removed the compounds overlapping with the training set , which led to 1347 compounds ( 230 positives and 1117 negatives ) as an external test set . we also merged the training with the test set and tried to generate a model based on the united data . the merged data set comprises 1904 compounds : 355 positives and 1549 negatives for cholestasis . here we must note that the total number of compounds in the merged data set is not the same as the sum of the compounds of training set and test set . the reason is that when removing the overlapping compounds between training set and test set , all of the compounds were removed from the test set , since we had already selected the final model for cholestasis . from those overlapping compounds , this did not matter so much , since they were totally removed from the test set . however , when merging the two data sets for modeling , we did not want to decide regarding the class of those compounds . for both data sets , several types of molecular descriptors have been calculated , such as all 192 2d moe descriptors , the 3d volsurf series of descriptors , as well as ecfps ( extended connectivity fingerprints ; ecfp6 ) , using rdkit ( http://www.rdkit.org/ ) . the list of the final descriptors used for the proposed model is given in the supporting information ( table s1 ) . in addition to this , predicted hepatic transporter inhibition profiles were also included in the list of descriptors . the transporters investigated comprise bsep , p - gp , bcrp , oatp1b1 , and oatp1b3 . in particular , for basolateral transporters we calculated the predictions for four in silico classification models built upon padel descriptors for oatp1b1 and oatp1b3 inhibition . for obtaining the predictions we use the models version implemented in etoxlab , an open source modeling framework for implementing predictive models . out of each model we use the sum of these binary scores , denoted sum binary score . the sum binary score can take values between 0 ( if all models predict the compound as negative ) and 4 ( if all models predict the compound as positive ) . for basolateral transporters a more thorough description of the transporters inhibition models is provided in the supporting information ( table s2 ) , where the size of the training set , the inhibition threshold of the training set , and the algorithm and performance of each individual model based on auc values are provided . the two - class classification models were built using the software package weka ( version 3.7.12 ) . we investigated the performance of several base classifiers , such as logistic regression , tree methods ( random forest and j48 tree ) , support vector machines ( smo in weka with polynomial , rbf , and puk kernels ) , nave bayes , and k - nearest neighbors . moreover , because the data set is slightly imbalanced , in order to equilibrate the effect of the majority class on model performance , we also applied the cost - sensitive meta - classifier metacost . the cost matrix applied corresponds to the imbalance ratio of the data ( 3:1 ) . additionally , several meta - classifiers were explored for attribute selection ( attributeselectedclassifier ) , as well as for improving the statistical performance , such as bagging and boosting . the models were originally validated via 10-fold cross validation , which is considered a quite trustworthy method of validation . the best models according to 10-fold cross - validation evaluation were further validated via using the data set by mulliner . subsequently , for the best obtained models , 50 iterations were performed by changing the cross - validation seed ( for splitting the data within cross validation ) and the respective performance parameters were calculated . in order to compare whether the inclusion of the transporters predictions in the descriptors set improves significantly the model s performance , a two - sample t test was performed in r. the statistics metrics taken into consideration were accuracy , sensitivity , specificity , matthews correlation coefficient ( mcc ) , area under the curve ( auc ) , precision and weighted average precision . apart from the conventional metrics of accuracy , sensitivity , and specificity , we also use auc , since it is a measure of the models capability to rank the predictions , while it decouples classifiers from class imbalance and error costs . moreover receiver operating characteristic ( roc ) graphs are very useful for visualization of the models results . the mcc value , considering its formula , takes into account all values of the confusion matrix : where , tp is true positives , fp is false positives , tn is true negatives , and fn is false negatives . thus , it is considered more balanced and informative than the column- or linewise metrics . weighted average precision is the average precision obtained for the two classes but weighted from the total number of instances of the classes . it is a quite helpful parameter in multiclass classification problems , as well as for imbalanced data sets where the number of negatives is greater than the number of positives . especially for the latter case , due to the definition of precision [ ppv = tp/(tp + fp ) ] , its value for the positive class would be low , which not necessarily means that the total performance of the model is bad . of course , since we are dealing with a toxicity classification problem , like cholestasis , the metrics that is of particular interest and that should by no means drop below 0.5 is sensitivity or true positive rate . in order to be confident regarding the validity of the models we used , we investigated the coverage of the transporters models for the cholestasis data . additionally , we checked how reliable the predictions of the cholestasis model for the cholestasis test set are . the applicability domain was checked on knime with the enalos nodes that compute the applicability domain on the basis of the euclidean distances . the number of compounds within the model s applicability domain for each model and for each cholestasis data set is provided in the supporting information ( table s3 ) . several combinations of descriptors and classifiers were investigated and the optimal classification model was selected on the basis of the results of 10-fold cross validation . with respect to the classifier , the best results were obtained using as base classifier ibk the k - nearest neighbors implementation in weka with k = 5 . the meta - classifier metacost was also applied , with the application of the cost matrix [ 0.0 , 1.0 ; 3.0 , 0.0 ] , i.e. weighting the minority class 3 times more than the majority class , in order to cope with the slightly imbalanced training set . 2d moe descriptors were performing better than fingerprints and/or volsurf descriptors , especially for sensitivity , mcc and auc . combining the volsurf descriptors with 2d moe descriptors also did not provide any significant improvement of the results . from the whole set of 2d moe descriptors we decided to use a subset of 93 interpretable descriptors that give almost the same performance compared to using all 2d moe descriptors . apart from the 93 2d descriptors , we also included the predicted transporter inhibition profiles . in order to assess the importance and significance of this additional information individually , we used them in different combinations : all transporters , only bsep , all transporters excluding either bsep , or p - gp , or bcrp , or the oatps . this led to in total seven models ( table 1 ) . it is interesting to mention that we also exchanged the training - test set roles between the two data sets and tried to generate a model for cholestasis based on the bigger data set from the work of mulliner et al . however , even though the results for 10-fold cross validation were equivalent to the model we propose ( generated on the compiled training set of 578 compounds ) , the results for the external validation of the 578 compounds were rather poor ( results not shown ) . furthermore , we tried to generate a cholestasis model on the merged data for the subset of 93 2d moe descriptors , with or without the transporters interaction profiles . interestingly , the k - nearest neighbor settings ( k = 5 ) , which gave quite satisfactory results for 10-fold cross validation while modeling either the training or the test set standalone , did not have the same effect for the united data . for the merged data set svm ( smo implementation in weka ) using a polynomial kernel , with exponent equal to 2 , performs better . the use of metacost with a cost matrix of [ 0.0 , 1.0 ; 5.0 , 0.0 ] , due to the new imbalance ratio of the data , is also necessary . additionally , under these settings , the performance of the model is significantly better when using the transporters predictions as additional descriptors . the obtained performance of this model , as well as the respective p - values of the performed two - sample paired t test out of 50 iterations , is presented in the supporting information ( table s4 ) . inspecting the obtained results in table 1 , it becomes obvious that the best settings for the model for 10-fold cross validation are achieved with the inclusion of all transporter inhibition predictions in the list of descriptors . nevertheless , this is not the case for the external validation , where including predicted inhibitor profiles for all transporters yields lower accuracy and specificity values , while sensitivity remains almost the same . interestingly , the use of bsep inhibition prediction stand - alone does not seem to be sufficient . there is a drop in the statistics especially for sensitivity in comparison to the use of the whole set of transporter predictions , both for 10-fold cross - validation and for the external test set . in order to assess if the predicted transporter inhibition profiles indeed statistically significantly improve the models , we performed 50 iterations of 10-fold cross validation followed by a two sample t test on the performance parameters . for this we used the models with 2d moe descriptors , 2d moe plus all transporters , 2d moe plus bsep , and 2d moe plus all transporters without bsep ( table 2 ) . analyzing the p - values for the pairwise comparisons ( supporting information table s5 ) the main conclusion is that indeed the use of liver transporter inhibition predictions contributes significantly to the models performance when compared to the use of only 2d physicochemical descriptors . interestingly , it is not only the bsep inhibition contribution , which matters . on the contrary , when only bsep predictions are used , there is only a slight increase in sensitivity and specificity of the model , while sensitivity decreases . the other way round , if all transporters predictions are used , sensitivity increases , but accuracy and specificity reach their peak only after the inclusion of bsep predictions . nevertheless , it does not contain all the important information , despite the fact that it is the most widely discussed transporter in literature with respect to cholestasis . a possible explanation for this interesting result could be the different thresholds required for bsep inhibition to cause cholestasis versus the threshold of the bsep inhibition model . for the bsep model , every compounds with an ic50 > 50 m was classified as noninhibitors , while ic50 < 10 m was the threshold for characterizing a compound as inhibitor . however , for the development of cholestasis , an ic50 < 300 m has been reported as sufficient . the other transporters included in our study are in general not well described in literature via experimental procedures , but they are rather pinpointed due to the fact that they are transporting bile salts or bile conjugates ( with the exception of p - gp , whose role is attributed mainly to drug thus , our study gives extra weight to literature indications concerning bcrp , p - gp , oatp1b1 , and 1b3 . it was quite curious that even though the transporters predictions significantly increase the performance both for the model built on the training set of 578 compounds and for the model trained with the merged data set of 1904 compounds ( but with a different base classifier ) ; this was not the case for the prediction on the test set . the performance of all statistics metrics decrease , when transporters predictions are used as descriptors . we can only speculate a different way of class assignment between the two data sets , since they are coming from different sources . another plausible explanation could be the fact that the external validation set had a contradiction rate of almost 20% regarding the class labels of those compounds shared with the training set ( 71 out of 419 shared compounds had contradictory class labels ) . we assume that this is due to the drawbacks of the toxicity reporting system : under - reporting , voluntarily basis , difficulties to obtain the data , which are often proprietary , as well as the lack of the prerequisite of a causal relationship between drug and adverse event . in any case , despite these contradictions between the training and the test set , the model retained its satisfactory performance . furthermore , we would like to mention that there is also experimental evidence for the implication of the basolateral efflux transporters mrp3 and mrp4 , as well as for the canalicular efflux transporter mrp2 . we are aware of this fact , but for these transporters there are currently not sufficient data available to develop high quality models that can be further used for contributing to our cholestasis model . additionally , we should pinpoint the fact that we are using predictions for the inhibition of transporters rather than real in vitro data . this could potentially accumulate some additional error in the final predictions of the cholestasis model . in any case , despite any deteriorating factors , our final in silico models for cholestasis were extensively validated with 10-fold cross validation and statistical tests . furthermore , the external validation set was of a significant size being even bigger than the training set . for both cases moreover , for both training sets ( regular and merged one ) there is a trend indicating the importance of transporter predictions in the development of cholestasis . the performance of the classification models from which we obtained the predictions provides us with enough confidence to use them in our input matrix . moreover , the percentage of the cholestasis data that are within the applicability domain of the transporters models ranges between 93.1% and 99.5% ( table s3 , supporting information ) , which is quite satisfactory and further enhances our confidence in using predicted transporter interaction profiles as descriptors . in this study we present a two - class classification model for the prediction of cholestasis ( or cholestatic dili ) based on a public data set of 578 compounds . the model is incorporating information both from 2d physicochemical descriptors , as well as predictions of inhibition of the hepatic transporters bsep , bcrp , p - gp , oatp1b1 , and oatp1b3 . the performance of the resulting model is rather satisfactory and is validated both via 50 iterations of different 10-fold cross validations , as well as an external test set of over 1300 compounds . our results demonstrate that adding transporter predictions as additional descriptors to the list of 2d physicochemical descriptors is improving model performance . this is in alignment with evidence from literature which shows that inhibition of selected hepatic transporters contributes to cholestasis . interestingly , the increase in model performance can not be attributed solely to bsep inhibition , which is the transporter that is most correlated to cholestasis in literature . the performance increases only when the whole list of transporter inhibition predictions is included . this result points toward a rather synergistic effect of several transporters , including the less elucidated role of oatps , bcrp , and p - gp in cholestasis . our study is the first of its kind regarding combining physicochemical descriptors and predicted transporter information in order to predict cholestasis . this was a double - blind randomized paralleled trial conducted at 74 sites in the u.s . and six sites in puerto rico ( nct00698230 ) . the study consisted of five periods : screening , metformin dose stabilization , 14-day placebo single - blind run - in , 12-week double - blind treatment , and 3-week off - treatment follow - up . the study was conducted pursuant to the declaration of helsinki and was approved by institutional review boards at participating sites . patients provided informed consent before screening . patients ( 1875 years ) with type 2 diabetes , a bmi between 25 and 45 kg / m , and a1c between 711% while taking metformin monotherapy at a stable dose for 10 weeks were eligible . exclusion criteria included a medical history of disorders involving glucocorticoid , mineralocorticoid , or androgen excess ; a history of type 1 diabetes or secondary forms of diabetes ; previous insulin therapy ; triglycerides > 500 mg / dl ; and treatment with any oral , systemic , topical , or inhaled glucocorticoids , thiazolidinediones , or exenatide within 3 months of screening . no inclusion criteria were specified for cholesterol or blood pressure and patients could enter the study on ( and maintain ) any hypolipidemic or antihypertensive regimen . patients were randomized equally to once - daily incb13739 ( 5 , 15 , 50 , 100 , or 200 mg ) or placebo . dose selection was based on phase 1 pharmacokinetic and pharmacodynamic data , with the goal of evaluating regimens that achieve different degrees of inhibition , from < 50 to > 90% , with the duration of inhibition varying across the five dose levels . patients with a fasting plasma glucose ( fpg ) > 270 mg / dl through week 8 or > 240 mg / dl subsequently were discontinued and offered rescue therapy . the primary end points were the change from baseline to week 12 compared with placebo in a1c , safety , and tolerability . secondary end points included the change from baseline to week 12 compared with placebo in fpg and lipid profiles and the proportion of patients achieving an a1c 7% at week 12 . tertiary end points included the change from baseline in homeostasis model assessment insulin resistance ( homa - ir ) , weight , blood pressure , and the proportion of patients meeting rescue therapy criteria . on - treatment study visits occurred at weeks 2 , 4 , 8 , and 12 and a follow - up visit at week 15 off treatment . monitoring for adverse events ( aes ) ( intensity , duration , outcome , and causality ) , physical examinations , vital signs , body weight and morphometrics , 12-lead electrocardiograms , and safety laboratory assessments including hematology , serum chemistry , and urinalysis were also performed . there were 40 patients per group completing week 12 who provided 90% power to detect a mean 0.6% difference in a1c between the 200-mg group and placebo assuming an emax dose - response model ( 13 ) with a half - maximal stimulation ( ed50 ) of 30 mg and an sd in a1c of 1.2% . this emax model is commonly used for phase 2 dose - ranging studies and was prespecified with the following optimal linear contrast : 0.45666 ( placebo ) , 0.31381 ( 5 mg ) , 0.12333 ( 15 mg ) , 0.168336 ( 50 mg ) , 0.312566 ( 100 mg ) , and 0.412901 ( 200 mg ) based on the half - maximal concentration ( ed50 ) = 30 mg assumption . two populations were prespecified : the evaluable analysis set was defined as all patients randomized who have completed the 12 weeks of study treatment with 80% compliance ; and the full analysis set was defined as all patients randomized who have taken at least one dose of study drug with any missing week 12 data imputed by last observation carried forward . the a1c and fpg end points were prespecified to be analyzed using the evaluable analysis set ; all other efficacy end points were prespecified to be analyzed using the full analysis set . for all end points , treatment effect was assessed using a linear model with treatment as the model factor and baseline as a covariate . on - treatment study visits occurred at weeks 2 , 4 , 8 , and 12 and a follow - up visit at week 15 off treatment . monitoring for adverse events ( aes ) ( intensity , duration , outcome , and causality ) , physical examinations , vital signs , body weight and morphometrics , 12-lead electrocardiograms , and safety laboratory assessments including hematology , serum chemistry , and urinalysis were also performed . there were 40 patients per group completing week 12 who provided 90% power to detect a mean 0.6% difference in a1c between the 200-mg group and placebo assuming an emax dose - response model ( 13 ) with a half - maximal stimulation ( ed50 ) of 30 mg and an sd in a1c of 1.2% . this emax model is commonly used for phase 2 dose - ranging studies and was prespecified with the following optimal linear contrast : 0.45666 ( placebo ) , 0.31381 ( 5 mg ) , 0.12333 ( 15 mg ) , 0.168336 ( 50 mg ) , 0.312566 ( 100 mg ) , and 0.412901 ( 200 mg ) based on the half - maximal concentration ( ed50 ) = 30 mg assumption . two populations were prespecified : the evaluable analysis set was defined as all patients randomized who have completed the 12 weeks of study treatment with 80% compliance ; and the full analysis set was defined as all patients randomized who have taken at least one dose of study drug with any missing week 12 data imputed by last observation carried forward . the a1c and fpg end points were prespecified to be analyzed using the evaluable analysis set ; all other efficacy end points were prespecified to be analyzed using the full analysis set . for all end points , treatment effect was assessed using a linear model with treatment as the model factor and baseline as a covariate . baseline assessments were performed in 302 patients who entered the treatment phase of the study , and 228 patients ( 75% ) completed the 12-week treatment period . the most common reasons for discontinuation were loss to follow - up ( 5% ) , withdrawal of consent ( 5% ) , lack of efficacy ( 4% ) , noncompliance with study procedures / medication ( 4% ) , and adverse events ( 4% ) , none of which related to the dose level of study medication . the clinical characteristics of the population at baseline were similar between treatment groups ( supplementary table 1 ) : the mean duration of diabetes was 6.2 years , bmi 32.4 kg / m , a1c 8.3% , and fpg 173 mg / dl . at week 12 , treatment with incb13739 resulted in a dose - dependent reduction in a1c ( pemax = 0.016 ; table 1 , fig . the placebo - adjusted least - squares ( ls ) mean difference from baseline in a1c reached statistical significance for the 100-mg ( 0.47% ; p < 0.05 ) and 200-mg ( 0.56% ; p a1c decreased compared with placebo in a time - dependent manner , reaching its maximum at week 12 ( fig . . a greater proportion of patients ( 25% ) randomized to 100 or 200 mg incb13739 achieved an a1c < 7% when compared with placebo ( 9.5% ) at week 12 . in a predefined subgroup analysis in patients with a baseline a1c 8% , the response to incb13739 was more pronounced , with the 50- , 100- , and 200-mg groups achieving a significant ( p < 0.05 ) change in a1c from baseline of 0.65 to 0.72% . the placebo - adjusted change in a1c for the 100- and 200-mg groups was greater in subjects with a baseline bmi > 30 kg / m ( 0.53% and 0.93% , respectively ) than in subjects with a baseline bmi 30 kg / m ( 0.35% and 0.17% , respectively ) . the number of patients requiring rescue therapy ( 12 ) did not differ significantly between treatment groups . fpg decreased in a dose- and time - dependent manner in the 100- and 200-mg treatment groups ( fig . 1c ) and reached statistical significance ( p < 0.01 ) from placebo in the 200-mg group with an ls mean difference of 24.1 mg / dl . a dose - dependent reduction in homa - ir was observed , reaching significance ( p < 0.05 ) in the 200-mg group with an ls mean difference of 1.32 ( 24% ) , suggesting an insulin - sensitizing mechanism of action . p < 0.01 , active vs. pbo . # p < 0.01 , week 12 vs. baseline . a : ls mean ( se ) change from baseline in a1c at week 12 . b : ls mean difference ( se ) from placebo in a1c from baseline to week 12 in the 100-mg ( ) and 200-mg ( ) treatment groups . c : ls mean difference ( se ) from placebo in fpg from baseline to week 12 in the 100- and 200-mg treatment groups . * p < 0.1 , p < 0.05 , p < 0.01 , active vs. placebo ( pbo ) . body weight decreased with incb13739 treatment , with statistical significance from baseline ( p < 0.05 ) achieved in the 15 ( 0.6 kg ) , 100 ( 1.1 kg ) , and 200 mg ( 0.9 kg ) treatment groups ( table 1 ) . plasma lipids and blood pressure were generally well controlled at baseline ( supplementary table 1 ) . treatment with incb13739 resulted in a modest dose - dependent ( ptrend = 0.026 ) decrease in total cholesterol , reaching a maximum of 7 mg / dl ( 3% ) from baseline in the 200-mg group ( table 1 ) . in a prespecified analysis , patients with adult treatment panel ( atp ) iii defined hyper - lipidemia ( total cholesterol > 200 mg / dl ; ldl cholesterol > 130 mg / dl ) or hyper - triglyceridemia ( > 200 mg / dl ) at baseline exhibited a greater improvement , reaching statistical significance ( p < 0.05 ) in the 100-mg group for all three lipid categories ( cholesterol 16 mg / dl , 6% ; ldl 17 mg / dl , 10% ; triglycerides 74 mg / dl , 16% ) . similar responses were observed in the 200-mg group , but these did not reach significance , possibly because of the smaller size of the subgroups . changes in hdl and free fatty acids were not significantly different between the treatment groups . treatment with incb13739 was well tolerated and aes were reported at similar frequencies across all treatment groups ( table 2 ) . one death occurred in the 200-mg group because of complications after a serious ae of acute ischemia of the lower extremities . this ae occurred 2 weeks after the last dose of study medication in a subject with preexisting congestive heart failure and aortic valvular disease . the death was due to cardiac arrest immediately after induction of anesthesia before bilateral iliofemoral embolectomy . the most frequent aes reported were typical for this population and did not exhibit dose dependence . there were four reports of nausea in the 200-mg group ( compared with one in the placebo group ) ; however , all of these resolved during continued dosing and three were categorized by the investigator as unrelated to study medication . there were no clinically relevant differences between treatment groups in electrocardiograms , hematology , serum chemistry , or urinalysis . end point endocrine assessments and safety summary endocrine data are week 12 ls mean sem unless otherwise noted . treatment emergent ae data are n ( % ) for all aes or for those occurring in at least 3% of patients . a4 , androstenedione ; d / c , discontinuation ; fai , free androgen index ; sae , serious adverse event ; t , testosterone . * determined by the investigator to be possibly , probably , or definitely drug related . the anticipated compensatory activation of the hypothalamic - pituitary - adrenal axis to overcome reduced cortisol regeneration on 11hsd1 inhibition was evaluated . incb13739 caused a dose - related increase in morning plasma acth and the acth - sensitive adrenocorticosteroid dehydroepiandrosterone sulfate ( dheas ) levels , although mean concentrations of both hormones remained within laboratory reference ranges ( table 2 ) . acth and dheas rises after incb13739 reached a plateau at week 4 ( + 102 and + 54% , respectively , versus + 19 and + 6% in the placebo group ) ; did not exhibit a further increase at week 12 , even in the 200-mg treatment group ( + 114% and + 55% , respectively ) ; and returned to baseline levels by the 3-week follow - up visit ( fig . morning plasma cortisol and evening salivary cortisol levels were unaltered by incb13739 at any dose ( fig . 2c ) , suggesting that the rise in acth was a compensatory response . change in acth , dheas , and cortisol . a : ls mean percent ( % ) change ( se ) from baseline in acth by treatment group and time on therapy . b : ls mean percent ( % ) change ( se ) from baseline in dheas by treatment group and time on therapy . c : ls mean ( se ) cortisol concentrations at week 12 in the morning ( plasma concentrations , , left axis ) or at night ( salivary concentrations , , right axis ) . incb13739 treatment resulted in a dose - related increase in morning fasting serum androstenedione ( a4 ) , although mean concentrations remained within the laboratory reference range ( table 2 ) . in males , there were no differences between treatment groups in total testosterone , sex hormone binding globulin ( shbg ) , or free androgen index ( fai ) . in females , total testosterone ( available at baseline and week 8) increased in a dose - dependent manner with mean concentrations within the laboratory reference range . maximal concentrations were observed in the 200-mg group ( 1.8 vs. 1.3 nmol / l in the placebo group ; p < 0.05 ) . these changes occurred alongside modest increases in shbg ( assessed at week 12 ) , apparent in the 50- and 100-mg groups ( p < 0.05 ) , but not the 200-mg group . importantly , there were no significant differences between treatment groups in calculated fai in females ( placebo = 6.9 ; incb13739 range = 5.78.2 ) . at week 12 , treatment with incb13739 resulted in a dose - dependent reduction in a1c ( pemax = 0.016 ; table 1 , fig . 1a ) . the placebo - adjusted least - squares ( ls ) mean difference from baseline in a1c reached statistical significance for the 100-mg ( 0.47% ; p < 0.05 ) and 200-mg ( 0.56% ; p < 0.01 ) groups . a1c decreased compared with placebo in a time - dependent manner , reaching its maximum at week 12 ( fig . 1b ) . a greater proportion of patients ( 25% ) randomized to 100 or 200 mg incb13739 achieved an a1c < 7% when compared with placebo ( 9.5% ) at week 12 . in a predefined subgroup analysis in patients with a baseline a1c 8% , the response to incb13739 was more pronounced , with the 50- , 100- , and 200-mg groups achieving a significant ( p < 0.05 ) change in a1c from baseline of 0.65 to 0.72% . the placebo - adjusted change in a1c for the 100- and 200-mg groups was greater in subjects with a baseline bmi > 30 kg / m ( 0.53% and 0.93% , respectively ) than in subjects with a baseline bmi 30 kg / m ( 0.35% and 0.17% , respectively ) . the number of patients requiring rescue therapy ( 12 ) did not differ significantly between treatment groups . fpg decreased in a dose- and time - dependent manner in the 100- and 200-mg treatment groups ( fig . 1c ) and reached statistical significance ( p < 0.01 ) from placebo in the 200-mg group with an ls mean difference of 24.1 mg / dl . a dose - dependent reduction in homa - ir was observed , reaching significance ( p < 0.05 ) in the 200-mg group with an ls mean difference of 1.32 ( 24% ) , suggesting an insulin - sensitizing mechanism of action . p < 0.01 , active vs. pbo . # p < 0.01 , week 12 vs. baseline . a : ls mean ( se ) change from baseline in a1c at week 12 . b : ls mean difference ( se ) from placebo in a1c from baseline to week 12 in the 100-mg ( ) and 200-mg ( ) treatment groups . c : ls mean difference ( se ) from placebo in fpg from baseline to week 12 in the 100- and 200-mg treatment groups . * p < 0.1 , p < 0.05 , p < 0.01 , active vs. placebo ( pbo ) . body weight decreased with incb13739 treatment , with statistical significance from baseline ( p < 0.05 ) achieved in the 15 ( 0.6 kg ) , 100 ( 1.1 kg ) , and 200 mg ( 0.9 kg ) treatment groups ( table 1 ) . plasma lipids and blood pressure were generally well controlled at baseline ( supplementary table 1 ) . treatment with incb13739 resulted in a modest dose - dependent ( ptrend = 0.026 ) decrease in total cholesterol , reaching a maximum of 7 mg / dl ( 3% ) from baseline in the 200-mg group ( table 1 ) . in a prespecified analysis , patients with adult treatment panel ( atp ) iii defined hyper - lipidemia ( total cholesterol > 200 mg / dl ; ldl cholesterol > 130 mg / dl ) or hyper - triglyceridemia ( > 200 mg / dl ) at baseline exhibited a greater improvement , reaching statistical significance ( p < 0.05 ) in the 100-mg group for all three lipid categories ( cholesterol 16 mg / dl , 6% ; ldl 17 mg / dl , 10% ; triglycerides 74 mg / dl , 16% ) . similar responses were observed in the 200-mg group , but these did not reach significance , possibly because of the smaller size of the subgroups . changes in hdl and free fatty acids were not significantly different between the treatment groups . treatment with incb13739 was well tolerated and aes were reported at similar frequencies across all treatment groups ( table 2 ) . no drug - related serious aes occurred in the trial . one death occurred in the 200-mg group because of complications after a serious ae of acute ischemia of the lower extremities . this ae occurred 2 weeks after the last dose of study medication in a subject with preexisting congestive heart failure and aortic valvular disease . the death was due to cardiac arrest immediately after induction of anesthesia before bilateral iliofemoral embolectomy . the most frequent aes reported were typical for this population and did not exhibit dose dependence . there were four reports of nausea in the 200-mg group ( compared with one in the placebo group ) ; however , all of these resolved during continued dosing and three were categorized by the investigator as unrelated to study medication . there were no clinically relevant differences between treatment groups in electrocardiograms , hematology , serum chemistry , or urinalysis . end point endocrine assessments and safety summary endocrine data are week 12 ls mean sem unless otherwise noted . treatment emergent ae data are n ( % ) for all aes or for those occurring in at least 3% of patients . a4 , androstenedione ; d / c , discontinuation ; fai , free androgen index ; sae , serious adverse event ; t , testosterone . * determined by the investigator to be possibly , probably , or definitely drug related . the anticipated compensatory activation of the hypothalamic - pituitary - adrenal axis to overcome reduced cortisol regeneration on 11hsd1 inhibition was evaluated . incb13739 caused a dose - related increase in morning plasma acth and the acth - sensitive adrenocorticosteroid dehydroepiandrosterone sulfate ( dheas ) levels , although mean concentrations of both hormones remained within laboratory reference ranges ( table 2 ) . acth and dheas rises after incb13739 reached a plateau at week 4 ( + 102 and + 54% , respectively , versus + 19 and + 6% in the placebo group ) ; did not exhibit a further increase at week 12 , even in the 200-mg treatment group ( + 114% and + 55% , respectively ) ; and returned to baseline levels by the 3-week follow - up visit ( fig . morning plasma cortisol and evening salivary cortisol levels were unaltered by incb13739 at any dose ( fig . 2c ) , suggesting that the rise in acth was a compensatory response . change in acth , dheas , and cortisol . a : ls mean percent ( % ) change ( se ) from baseline in acth by treatment group and time on therapy . b : ls mean percent ( % ) change ( se ) from baseline in dheas by treatment group and time on therapy . c : ls mean ( se ) cortisol concentrations at week 12 in the morning ( plasma concentrations , , left axis ) or at night ( salivary concentrations , , right axis ) . incb13739 treatment resulted in a dose - related increase in morning fasting serum androstenedione ( a4 ) , although mean concentrations remained within the laboratory reference range ( table 2 ) . in males , there were no differences between treatment groups in total testosterone , sex hormone binding globulin ( shbg ) , or free androgen index ( fai ) . in females , total testosterone ( available at baseline and week 8) increased in a dose - dependent manner with mean concentrations within the laboratory reference range . maximal concentrations were observed in the 200-mg group ( 1.8 vs. 1.3 nmol / l in the placebo group ; p < 0.05 ) . these changes occurred alongside modest increases in shbg ( assessed at week 12 ) , apparent in the 50- and 100-mg groups ( p < 0.05 ) , but not the 200-mg group . importantly , there were no significant differences between treatment groups in calculated fai in females ( placebo = 6.9 ; incb13739 range = 5.78.2 ) . the results from this study indicate , for the first time , that decreasing local cortisol exposure through 11hsd1 inhibition improves hyperglycemia over 12 weeks in patients with type 2 diabetes . the addition of once - daily incb13739 in patients inadequately controlled with metformin significantly reduced a1c , fpg , and homa - ir . these effects were dose dependent , and the greatest improvements were achieved at the highest dose administered ( 200 mg ) , with evidence for a more profound a1c reduction in subjects with a bmi > 30 kg / m , compatible with elevated 11hsd1 in adipose tissue in obesity . preliminary data from pharmacokinetic analyses ( data not shown ) indicate that the 100- and 200-mg groups achieved , 4 h after administration , mean free drug exposures that reached 100 mg or exceeded 200 mg , the concentrations required to inhibit 90% of the enzyme activity in cellular assays ; however , only the 200-mg group retained such a mean exposure at the end of the dosing interval . thus , glycemic efficacy may be associated with a high degree of enzyme inhibition , and it is possible that greater glycemic improvement might be achieved with increased dose levels or frequency of administration . plasma lipids were generally well controlled in this population , and 30% of patients were receiving lipid - lowering medications . incb13739 treatment resulted in a dose - dependent reduction in total cholesterol , and while of modest magnitude , these changes also associated with directional beneficial trends in ldl cholesterol and triglycerides . of interest , patients who met atp iii criteria for borderline high ldl cholesterol ( > 130 mg / dl ) , total cholesterol ( > 200 mg / dl ) , or hyper - triglyceridemia ( > 200 mg / dl ) exhibited a larger improvement in all three lipid parameters . the magnitude of effect was equivalent in the 100- and 200-mg groups , reaching statistical significance for the 100-mg group , which had the largest subgroup size . incb13739 treatment resulted in a dose - dependent modest decrease in body weight of 1 kg at the highest dose studied . this change was time dependent and did not plateau over the 12-week treatment period ( data not shown ) . the thiazolidinedione insulin sensitizers increase body weight through adipocyte differentiation ( 14,15 ) . as cortisol can drive adipocyte differentiation and expansion ( 16 ) , it is possible that attenuating cortisol signaling in adipose may decrease adipocyte size . this has been reported in preclinical models with an 11hsd1 inhibitor ( 17 ) and suggests the potential for positive effects of incb13739 on total body weight and/or regional adiposity with longer exposure . incb13739 was well tolerated at all dose levels , and there were no differences in ae frequency relative to placebo nor were there any apparent dose - dependent changes in aes . while 11hsd1 is not involved in adrenal cortisol biosynthesis , 11hsd1 activity within the splanchnic bed does contribute 25% of total cortisol production ( 18 ) . an expected consequence of 11hsd1 inhibition is increased clearance of cortisol and compensatory hypothalamic - pituitary - adrenal axis activation to maintain blood cortisol concentrations . incb13739 treatment did result in a dose - related increase in acth levels that was generally within the normal reference range . the acth response reached a plateau with the 50-mg dose at week 4 , suggesting that the maximal response to incb13739 had been realized . this plateau in acth and its rapid return to baseline levels after cessation of therapy are consistent with an adaptive endocrine process driven by reversible 11hsd1 inhibition . these data indicate normal hypothalamic - pituitary - adrenal axis function after 12 weeks of incb13739 therapy that adjusted appropriately to the inhibition of 11hsd1 activity to maintain basal cortisol homeostasis . the leftward shift in the acth dose relationship relative to efficacy might reflect a greater contribution of hepatic 11hsd1 inhibition to splanchnic cortisol reactivation . aldosterone and renin were unaltered by incb13739 treatment ( table 2 ) , and serum electrolytes were unchanged ( supplementary table 2 ) . modest elevations in the androgenic precursors dheas and a4 paralleled changes in acth . like acth , these changes were generally within the reference range , plateaued with respect to both dose and time , and were reversed at follow - up . the highest concentration of dheas observed in this study ( 13.2 mol / l in males and females ) is equivalent to levels observed after 50 mg / day dehydroepiandrosterone supplement use ( 19 ) . in men , there was no change in plasma testosterone , shbg , or fai after incb13739 treatment , consistent with the testes being the main source of androgens . in females , a modest rise in total testosterone at week 8 was observed that was paralleled by a rise in shbg such that the resulting fai calculation was not significantly different in any incb13739 group compared with placebo or baseline levels . shbg is known to increase in response to improved insulin sensitivity ( 20 ) , and whether the changes observed in this study reflect this or result from more complex endocrine adaptation to small changes in total testosterone are unknown . importantly , fai is an accepted surrogate in clinical practice for free testosterone and a marker of biologic androgen activity in women ( 21 ) . no signs or symptoms of androgen excess were observed , and longer - term studies will be required to ascertain the clinical relevance of the small androgen changes observed . in summary , in patients with type 2 diabetes who had inadequate glycemic control with metformin alone , the addition of once - daily incb13739 was well tolerated and resulted in significant improvements in a1c , fpg , and homa - ir . incb13739 treatment decreased body weight and improved cholesterol and triglycerides in patients with hyper - lipidemia at baseline . 11hsd1 inhibition offers a new potential approach to control glucose and cardiovascular risk factors in type 2 diabetes .

cholestasis represents one out of three types of drug induced liver injury ( dili ) , which comprises a major challenge in drug development . in this study we applied a two - class classification scheme based on k - nearest neighbors in order to predict cholestasis , using a set of 93 two - dimensional ( 2d ) physicochemical descriptors and predictions of selected hepatic transporters inhibition ( bsep , bcrp , p - gp , oatp1b1 , and oatp1b3 ) . in order to assess the potential contribution of transporter inhibition , we compared whether the inclusion of the transporters inhibition predictions contributes to a significant increase in model performance in comparison to the plain use of the 93 2d physicochemical descriptors . our findings were in agreement with literature findings , indicating a contribution not only from bsep inhibition but a rather synergistic effect deriving from the whole set of transporters . the final optimal model was validated via both 10-fold cross validation and external validation . it performs quite satisfactorily resulting in 0.686 0.013 for accuracy and 0.722 0.014 for area under the receiver operating characteristic curve ( auc ) for 10-fold cross - validation ( mean standard deviation from 50 iterations ) . objective11--hydroxysteroid dehydrogenase type 1 ( 11hsd1 ) converts inactive cortisone into active cortisol , thereby amplifying intracellular glucocorticoid action . the efficacy and safety of the 11hsd1 inhibitor incb13739 were assessed when added to ongoing metformin monotherapy in patients with type 2 diabetes exhibiting inadequate glycemic control ( a1c 711%).research design and methodsthis double - blind placebo - controlled paralleled study randomized 302 patients with type 2 diabetes ( mean a1c 8.3% ) on metformin monotherapy ( mean 1.5 g / day ) to receive one of five incb13739 doses or placebo once daily for 12 weeks . the primary end point was the change in a1c at study end . other end points included changes in fasting glucose , lipids , weight , adverse events , and safety.resultsafter 12 weeks , 200 mg of incb13739 resulted in significant reductions in a1c ( 0.6% ) , fasting plasma glucose ( 24 mg / dl ) , and homeostasis model assessment insulin resistance ( homa - ir ) ( 24% ) compared with placebo . total cholesterol , ldl cholesterol , and triglycerides were all significantly decreased in hyperlipidemic patients . body weight decreased relative to placebo after incb13739 therapy . a reversible dose - dependent elevation in adrenocorticotrophic hormone , generally within the normal reference range , was observed . basal cortisol homeostasis , testosterone in men , and free androgen index in women were unchanged by incb13739 . adverse events were similar across all treatment groups.conclusionsincb13739 added to ongoing metformin therapy was efficacious and well tolerated in patients with type 2 diabetes who had inadequate glycemic control with metformin alone . 11hsd1 inhibition offers a new potential approach to control glucose and cardiovascular risk factors in type 2 diabetes .

however , it is unclear whether to perform reoperation in asymptomatic patients with low transprosthetic gradient . especially , in patients with depressed left ventricular function , it is important to clarify which the main cause of left ventricular dysfunction is structural valve deterioration or other factors such as cardiomyopathy . this case is a 78-year - old male who underwent aortic valve replacement with a 23 mm carpentier - edwards perimount pericardial bioprosthesis ( edwards lifesciences , irvine , ca , usa ) for aortic valve stenosis 7 years ago at another hospital . pulmonary sarcoidosis was diagnosed in his 50s , and cardiac sarcoidosis was diagnosed by a myocardial biopsy at the time of the previous cardiac surgery . he had a past history of thyroid cancer , and his thyroids and parathyroids were resected at the age of 45 , and then after that , he had been prescribed with levothyroxine and alfacalcidol . the echocardiography 6 years after the previous operation showed a normal opening of his prosthesis with an effective orifice area index of 0.62 cm / m , a mean pressure gradient of 15 mmhg , and mildly reduced left ventricular function with an ejection fraction of 43% . although he was asymptomatic , his transthoracic echocardiography 1 year later revealed the restricted motion of the prosthetic valve leaflets , with an effective orifice area index of 0.28 cm / m and a mean pressure gradient of 28 mmhg . it also revealed depressed left ventricular function with an ejection fraction of 37% , moderate mitral insufficiency , and severe tricuspid insufficiency . considering comparably early deterioration of his bioprosthesis valve and left ventricular function , we decided to perform a dobutamine stress echocardiography to differentiate structural valve deterioration from pseudo - aortic stenosis due to cardiac sarcoidosis . at the baseline , the aortic valve peak velocity and the stroke volume index were 3.8 m / s and 31 ml / m . at a dose of 10 g / kg / min of dobutamine infusion , they increased to 4.4 m / s and 41 ml / m , respectively , and the mean pressure gradient across the bioprosthesis increased to 47 mmhg , as the effective orifice area index remained small at 0.29 cm / m . therefore , he was diagnosed as having structural valve deterioration of bioprosthesis at aortic position . he underwent redo aortic valve replacement with a 20 mm ats standard mechanical valve ( ats medical , minneapolis , minnesota , usa ) , mitral annuloplasty with a 26 mm carpentier - edwards physio ii annuloplasty ring ( edwards lifesciences , irvine , ca , usa ) , and tricuspid annuloplasty with a 28 mm edwards mc3 tricuspid annuloplasty ring ( edwards lifesciences , irvine , ca , usa ) . a transthoracic echocardiography at 6 months after the reoperation showed a recovery of left ventricular function with ejection fraction of 49% . pathologic examination of the explanted prosthesis showed calcified deposit and desmoplasia in all the leaflets without an evidence of infiltration of monocyte [ figure 1 ] . we reported a rare case of low transprosthetic gradient structural valve deterioration at aortic position in a patient with cardiac sarcoidosis . the principal manifestations of cardiac sarcoidosis are conduction abnormalities , ventricular arrhythmias , and heart failure . considering early bioprosthetic deterioration in a patient with cardiac sarcoidosis , there were three challenging points to be discussed in this case . first , it was difficult to diagnose which the main cause of depressed left ventricular function was structural valve deterioration or cardiac sarcoidosis . third , the optimal strategy for this case including the type of surgery and prosthesis was worth debating . this case was low - gradient aortic stenosis , which was defined as an aortic valve area < 1.0 cm and a mean transvalvular gradient < 40 mmhg . one of the diagnostic challenges in low - gradient aortic stenosis with depressed left ventricular function is to differentiate true severe aortic stenosis from pseudo - aortic stenosis . in this case , if the cause of depressed left ventricular function was structural valve deterioration , redo aortic valve replacement would be strongly recommended . on the other hand , if his depressed left ventricular function was the clinical manifestation of the cardiac sarcoidosis , the benefit of reoperation on the ventricular reverse remodeling was uncertain . a dobutamine stress echocardiography is useful to differentiate true severe aortic stenosis from pseudo - aortic stenosis in case of native aortic valve . it remains unclear whether it is also useful in case of bioprosthetic valve deterioration , but it could lead us to the diagnosis of true structural valve deterioration . only 7 years had passed until the reoperation for structural valve deterioration in this case . reported several risk factors of early bioprosthetic deterioration , but this case did not have any of these factors . skolnick et al . reported the association between osteoporosis and decreased progression of aortic stenosis , and speculated inhibition of valvular calcification might be due to alterations in levels of vitamin d and parathyroid hormone . on the other hand , it is reported chronic secondary hyperparathyroidism in renal failure is associated with aortic calcification . in patients with sarcoidosis , therefore , the possible cause of early bioprosthesis deterioration in this case might be alfacalcidol , an analog of vitamin d or parathyroid hormone - related protein due to sarcoidosis . the patient was asymptomatic , but it was reasonable to perform reoperation because the structural valve deterioration caused the left ventricular dysfunction . in this case , we decided to perform surgical aortic valve replacement with mechanical valve considering his early bioprosthetic valve deterioration and the possibility of the need for another redo aortic valve replacement . however , it might be another option to perform surgical aortic valve replacement with bioprosthetic valve and transcatheter valve - in - valve procedure if necessary in the future . future studies are warranted to determine what strategies about the optimal type of surgery and valve selection are reasonable . periodontitis is a chronic inflammatory disease of supporting tissues of the teeth in response to noxious stimuli whether mechanical , chemical or infectious , resulting in progressive destruction of periodontal apparatus , hence leading to pocket formation , recession or both . the tissue destruction in periodontitis has been attributed to the production of enzymes and reactive oxygen species ( ros ) by polymorphonuclear cells and other cells . it has been demonstrated that patients with periodontitis have higher oxidative dna and lipid damage biomarkers and lower antioxidant ( ao ) enzymatic activities in saliva than healthy subjects . there are a variety of defense mechanisms in the body to combat excessive ros production , and aos are one among these . aos are those substances when present at a low concentration compared to those of an oxidizable substrate significantly delays or prevents oxidation of the substrate . superoxide and hydrogen peroxide are the main oxidative species produced , which are either enzymatically eliminated by preventive aos , or metal ions are sequestrated , hence preventing fenton reactions and subsequent hydroxyl radical formation e.g. catalase , glutathione ( gsh ) peroxidase , 7 s - transferase . interception involves scavenging / chain breaking aos which inhibit chain initiation , chain propagation and also lipid peroxidation e.g. lipophilic substances such as ubiquinol , vitamin a , vitamin e , carotenoids and hydrophilic substances like uric acid , ascorbic acid , albumin , and bilirubin . at repair level various aos function by repairing the damaged and reconstituting membranes . when the level of ros increases intracellularly , the cellular ao defenses are insufficient to maintain these harmful molecules , this condition is generally referred to as oxidative stress . the concept of oxidative stress dates back to 1986 and was elaborated as the relation between free radicals and disease . gsh is a ubiquitous tripeptide made from the combination of three amino acids , that is , cysteine , glutamate , and glycine . it is a low molecular weight thiol ( up to 510 mm ) present in the cell and existing in two forms which are oxidized glutathione ( gssg ) and reduced gsh forms . out of the total gsh present in the body , the reduced gsh constitutes about 90% , and gssg is 10% . for survival of cell , oxidative damage results when the there is a deficiency of reduced gsh in the cell , which puts it at risk for oxidative damage . anti - oxidant function [ figure 1]detoxification functionimmune function . anti - oxidant function [ figure 1 ] detoxification function schematic representation of the biochemical interactions between neutrophil superoxide and glutathione the purpose of this study is to compare the levels of gsh , both oxidized and reduced forms in patients with and without chronic periodontitis in gingival crevicular fluid ( gcf ) . this study was a 6 months randomized case control study , which was conducted at the department of periodontology . an approval for the study was obtained from the ethical committee , and written informed consent was obtained before enrolling the subjects for the study . the study enrolled 40 patients , which included patients with chronic periodontitis n = 20 ( test group ) and patients without chronic periodontitis n = 20 ( control group ) . subjects in the test group were selected on the basis of clinical criteria of having at least two nonadjacent sites per quadrant with probing pocket depths 5 mm , along with bleeding on probing and demonstrable radiographic bone loss . control patients did not show evidence of attachment loss or probing pocket depths 3 mm and bleeding scores were below 10% . the subjects with a history of taking vitamin supplements , anti - inflammatory or antibiotic medication in the preceding 3 months and pregnant ladies and patient with special dietary needs were not included in the study . after enrollment , baseline gcf samples were collected , before recording clinical measures which included silness and loe plaque index , loe and silness gingival index , muhlemann and son sulcus bleeding index and probing pocket depth . after data collection , chronic periodontitis patients ( test group ) underwent scaling and root planing and oral hygiene instruction which included brushing technique , dental flossing were reinforced at baseline . subjects were then recalled at 1-month , 3 months and 6 months posttherapy for recording clinical and biochemical parameters . gcf samples were collected from mesiobuccal / distolingual sites on any teeth in the maxillary quadrant ( test and control group ) using microcapillary pipettes . gcf samples were then immediately transferred to top sealed cuvettes to prevent oxidation of aos present in it . all the results of this study were obtained by comparing intragroup and intergroup parameters , at various designated phases at baseline , 1 month , 3 month and 6 month for test over control groups using paired student 's t - test using software computer software spss version 20 ( ibm spss inc , chicago , il , usa ) . this study was a 6 months randomized case control study , which was conducted at the department of periodontology . an approval for the study was obtained from the ethical committee , and written informed consent was obtained before enrolling the subjects for the study . the study enrolled 40 patients , which included patients with chronic periodontitis n = 20 ( test group ) and patients without chronic periodontitis n = 20 ( control group ) . subjects in the test group were selected on the basis of clinical criteria of having at least two nonadjacent sites per quadrant with probing pocket depths 5 mm , along with bleeding on probing and demonstrable radiographic bone loss . control patients did not show evidence of attachment loss or probing pocket depths 3 mm and bleeding scores were below 10% . the subjects with a history of taking vitamin supplements , anti - inflammatory or antibiotic medication in the preceding 3 months and pregnant ladies and patient with special dietary needs were not included in the study . after enrollment , baseline gcf samples were collected , before recording clinical measures which included silness and loe plaque index , loe and silness gingival index , muhlemann and son sulcus bleeding index and probing pocket depth . after data collection , chronic periodontitis patients ( test group ) underwent scaling and root planing and oral hygiene instruction which included brushing technique , dental flossing were reinforced at baseline . subjects were then recalled at 1-month , 3 months and 6 months posttherapy for recording clinical and biochemical parameters . gcf samples were collected from mesiobuccal / distolingual sites on any teeth in the maxillary quadrant ( test and control group ) using microcapillary pipettes . gcf samples were then immediately transferred to top sealed cuvettes to prevent oxidation of aos present in it . all the results of this study were obtained by comparing intragroup and intergroup parameters , at various designated phases at baseline , 1 month , 3 month and 6 month for test over control groups using paired student 's t - test using software computer software spss version 20 ( ibm spss inc , chicago , il , usa ) . the nonsurgical periodontal therapy provided to the test group resulted in observed reductions of whole mouth mean plaque scores ( p < 0.001 ) , mean gingival scores , and mean sulcus bleeding index scores in the group ( p < 0.001 ) which in turn showed reduced inflammation as compared to control group [ tables 16 ] . intra - group comparison of mean plaque scores and sd in test group intra - group comparison of mean gingival index scores and sd in test group intragroup comparison of mean sbi scores and sd in test group intragroup comparison of mean plaque scores and sd in control group intragroup comparison of mean gingival index scores and sd in control group intragroup comparison of mean sulcus bleeding index score and sd in control group in all groups , mean reduced gsh and gssg levels were detected in the millimolar range ( range : 0.305.14 mm [ table 7 ] ) . lower levels of gsh and gssg concentrations were reported in the gcf from chronic periodontitis patients before and after treatment , compared with those detected in nonchronic periodontitis subjects ( control group ) [ table 8 ] . mean ratio of reduced glutathione and oxidized glutathione and sd in test group intergroup comparison between various parameters used in study between test and control group comparative analysis of difference in mean gsh and gssg value for test group was done at different time intervals , namely , baseline 1-month , baseline - 3 months , baseline - 6 months , 13 months and 16 months . the differences were observed to be statistically significant ( p < 0.001 ) at a confidence interval of 95% , but statistically nonsignificant at the 36 months interval [ tables 9 and 10 ] . comparison of mean gsh and gssg score with a standard deviation between test and control group is depicted [ figures 2 and 3 ] gsh and gssg value for control group were depicted at different time intervals , namely , baseline 1 month , baseline - 3 months , baseline - 6 months , 13 months and 16 months in tables 11 and 12 . intragroup comparison of mean reduced glutathione level and sd in test group intragroup comparison of mean oxidized glutathione level and sd in test group reduced glutathione levels at different time intervals for test and control groups oxidized glutathione levels at different time intervals for test and control groups intragroup comparison of mean reduced glutathione level and sd in control group intragroup comparison of mean oxidized glutathione level and sd in control group ratio of reduced gsh and gssg for test group at different time interval during the study was reported to be statistically significant ( p < 0.001 ) at confidence interval of 95% at baseline 1-month , baseline - 3 months , baseline - 6 months , 13 months , 16 months [ table 7 ] . comparison of the mean ratio of reduced and oxidized gsh ( gsh : gssg ) score with a standard deviation between test and control group is depicted in figure 4 . this improvement in the gsh : gssg ratio after treatment was as a result of an increase in levels of reduced gsh and a reduction in levels of oxidized gsh . ratio of reduced to oxidized glutathione levels at different time intervals for test and control groups intragroup comparison of mean ratio of reduced glutathione and oxidized glutathione and sd in control group the nonsurgical periodontal therapy provided to the test group resulted in observed reductions of whole mouth mean plaque scores ( p < 0.001 ) , mean gingival scores , and mean sulcus bleeding index scores in the group ( p < 0.001 ) which in turn showed reduced inflammation as compared to control group [ tables 16 ] . intra - group comparison of mean plaque scores and sd in test group intra - group comparison of mean gingival index scores and sd in test group intragroup comparison of mean sbi scores and sd in test group intragroup comparison of mean plaque scores and sd in control group intragroup comparison of mean gingival index scores and sd in control group intragroup comparison of mean sulcus bleeding index score and sd in control group in all groups , mean reduced gsh and gssg levels were detected in the millimolar range ( range : 0.305.14 mm [ table 7 ] ) . lower levels of gsh and gssg concentrations were reported in the gcf from chronic periodontitis patients before and after treatment , compared with those detected in nonchronic periodontitis subjects ( control group ) [ table 8 ] . mean ratio of reduced glutathione and oxidized glutathione and sd in test group intergroup comparison between various parameters used in study between test and control group comparative analysis of difference in mean gsh and gssg value for test group was done at different time intervals , namely , baseline 1-month , baseline - 3 months , baseline - 6 months , 13 months and 16 months . the differences were observed to be statistically significant ( p < 0.001 ) at a confidence interval of 95% , but statistically nonsignificant at the 36 months interval [ tables 9 and 10 ] . comparison of mean gsh and gssg score with a standard deviation between test and control group is depicted [ figures 2 and 3 ] gsh and gssg value for control group were depicted at different time intervals , namely , baseline 1 month , baseline - 3 months , baseline - 6 months , 13 months and 16 months in tables 11 and 12 . intragroup comparison of mean reduced glutathione level and sd in test group intragroup comparison of mean oxidized glutathione level and sd in test group reduced glutathione levels at different time intervals for test and control groups oxidized glutathione levels at different time intervals for test and control groups intragroup comparison of mean reduced glutathione level and sd in control group intragroup comparison of mean oxidized glutathione level and sd in control group ratio of reduced gsh and gssg for test group at different time interval during the study was reported to be statistically significant ( p < 0.001 ) at confidence interval of 95% at baseline 1-month , baseline - 3 months , baseline - 6 months , 13 months , 16 months [ table 7 ] . comparison of the mean ratio of reduced and oxidized gsh ( gsh : gssg ) score with a standard deviation between test and control group is depicted in figure 4 . this improvement in the gsh : gssg ratio after treatment was as a result of an increase in levels of reduced gsh and a reduction in levels of oxidized gsh . ratio of reduced to oxidized glutathione levels at different time intervals for test and control groups intragroup comparison of mean ratio of reduced glutathione and oxidized glutathione and sd in control group the present data confirm the study conducted by chapple et al . , 2002 suggesting that a millimolar concentration of gsh was present in gcf , which decreases during periodontitis . the reduction in gsh levels is suggestive of its protective role for vital cells and tissue structures from host - derived free radicals . due to the conversion of gsh to gssg , there is a reduction in gsh levels at inflamed tissue sites as also seen in this study . similarly , the reduced gsh : gssg ratio at baseline was due to reduced levels of gsh as well as increased accumulation of gssg . these results were consistent with findings reported in earlier studies demonstrating a reduction in total antioxidant capacity of saliva in patients with chronic periodontitis along with reduced levels of gsh . in this study , a significant increase in gsh levels along with improvement in gsh : gssg ratio at 1-month was observed following nonsurgical periodontal therapy viz . , scaling and root planing , which is consistent with the observations made by several others studies . these results were suggestive of an improvement in ao status and reduction in oxidative stress following nonsurgical periodontal therapy . other possibilities for the improvement seen following nonsurgical therapy is the reduction in proteolytic activity of microorganisms and also reductions in inflammation posttherapy , thereby lowering levels of oxidants in gcf , both exogenously and endogenously . the improvement was seen in gsh : gssg ratio at 3 months is due to relatively long duration taken by subgingival microflora to reestablish , hence their reduced activity . the preliminary findings of studies stated that millimolar concentrations of gsh is present in gcf gets significantly reduced in chronic periodontitis patients . in addition , this study demonstrated that successful , nonsurgical periodontal therapy does restore the redox balance ( gsh : gssg ) till 3 months , but at 6 months no significant improvement in either gsh or gsh : gssg ratio was noted . thus after 3 months thorough oral hygiene practice is not self - sufficient for restoring the gsh levels and gsh : gssg ratio to health , this implies that there is a reduced potential of periodontal tissues against ros activity in chronic periodontitis patients , even after successful nonsurgical therapy . such findings have opened up the potential of using pharmacological agents to elevate buffering capacity within tissues by elevating gsh levels , e.g. use of the gsh promoting drug n - acetyl cysteine . anti - inflammatory tissue redox state can be created by supplementing the individuals with micronutrients for elevating gsh concentrations . for various chronic inflammatory diseases that are associated with redox imbalance , ao supplementation duration of the study , the reliability of the spectroscopic method used for gsh estimation and method of gcf collection . this study opens up scope for various future studies dealing with the role of gsh as biological diagnostic disease marker , ao , detoxifier , and immune modulator . long - term studies can be undertaken to evaluate the efficacy of novel therapeutic approaches for improving the buffering capacity within periodontal tissues by elevating the gsh levels . duration of the study , the reliability of the spectroscopic method used for gsh estimation and method of gcf collection . this study opens up scope for various future studies dealing with the role of gsh as biological diagnostic disease marker , ao , detoxifier , and immune modulator . long - term studies can be undertaken to evaluate the efficacy of novel therapeutic approaches for improving the buffering capacity within periodontal tissues by elevating the gsh levels . the study concluded that at baseline due to prevailing oxidative stress the levels of reduced gsh were reduced in patients with chronic periodontitis . following nonsurgical therapy the levels of gsh were improved at 1-month and 3 months . at 6 months recall following nonsurgical therapy , an adjunctive use of micronutritional supplements to boost ao concentration in tissues by preserving gsh or by elevating its level at the inflamed sites is recommended .

we report a case of structural valve deterioration , which occurred 7 years after aortic valve replacement in a 78-year - old male with cardiac sarcoidosis . his echocardiography showed low transprosthetic valve gradient and depressed left ventricular function . a dobutamine stress echocardiography was performed to identify his pathophysiology , and it revealed that his depressed left ventricular function was not due to cardiac sarcoidosis but to structural valve deterioration . reoperation for structural valve deterioration was performed , and his left ventricular function recovered . objective : to compare the levels of glutathione ( gsh ) , both oxidized and reduced forms in patients with and without chronic periodontitis in gingival crevicular fluid ( gcf).materials and methods : twenty gcf samples from maxillary quadrants were collected using capillary micropipettes from the chronic periodontitis patients ( test group ) at baseline before treatment , at 1-month , 3 months , and 6 months after scaling and root planing and samples from 20 patients without chronic periodontitis ( control group ) from maxillary quadrants were also collected . gsh , oxidized glutathione ( gssg ) levels and gsh : gssg ratios were determined using the spectrophotometric method.statistical analysis : results were concluded for the test over control groups using paired student 's t-test.results:lower concentrations of gsh ( p < 0.001 ) and gssg ( p < 0.001 ) were detected in gcf in patients with chronic periodontitis ( test group ) than patients without chronic periodontitis ( control group ) at baseline . treatment had a significant effect in improving the gsh and reducing gssg levels postscaling and root planing at 1-month and 3 months but not significant effect at 6 months . scaling and root planing increased the gsh : gssg ratio ( p < 0.001 ) in the test group as compared to the control group ( p < 0.001).conclusions : the concentrations of gsh within gcf are reduced in chronic periodontitis patients . scaling and root planing ( nonsurgical therapy ) restores gsh concentration in gcf post 1-month and 3 months along with redox balance ( gsh : gssg ratio ) , but at 6 months the balance is not maintained . adjunctive use of micronutritional supplements to boost antioxidant concentration in tissues by preserving gsh or by elevating its level at the inflamed sites is recommended , as nonsurgical periodontal therapy alone is not able to maintain redox balance for longer duration .

breast carcinoma is the commonest malignant tumor and the leading cause of cancer death in women , with more than one million new cases occurring worldwide annually . it is well known that proto - oncogenes and tumor suppressor genes are two types of genes that play a key role in the regulation of cell growth and differentiation . hence , any alteration in one or more of these genes appears to play an important role in the pathogenesis of most human malignancies . her-2/neu proto - oncogene amplification and/or overexpression is one of the most important alterations seen in breast cancer . the her-2/neu proto - oncogene ( also called c - erbb2 ) is located on chromosome 17q11[35 ] which encodes for p185 , a transmembrane glycoprotein with tyrosine kinase activity that belongs to the family of epidermal growth factor receptors . her2/neu proto - oncogene is amplified and/or overexpressed in approximately 2530% of invasive breast cancers . an association has been found to exist between amplification and/or overexpression of her-2/neu and a wide variety of different clinical and pathological features of breast carcinoma . from a clinical point of view , her-2/neu receptor has become an important target for antibody - based therapy with trastuzumab ( herceptin ) . in iraq , where the population was exposed to high levels of depleted uranium following the first and second gulf wars , breast cancer is the most common tumor type in females . over the last ten years , there has been a three - fold increase in the incidence of breast cancer , with most of this increase being attributed to a particularly aggressive type of the cancer . this finding inspired an investigation of her2/neu expression in a series of iraqi women with breast cancer to see whether any differences in her2/neu expression , in correlation with tumor characteristics , could be found in a population exposed to depleted uranium . ninety specimens of formalin - fixed , paraffin embedded breast cancer tissue , collected from breast cancer patients over a period from september 2007 to june 2008 were included in this study . all cases were collected from major hospitals and private laboratories in kufa district area ( located in the middle of iraq ) . the age range of patients was 21 to 70 years , with a mean age of 48.8 years . a group of 25 patients with benign breast lesions ( fibroadenoma ) was included as a comparative group and 20 normal breast tissue sections were included as controls . confirmation of histopathological diagnosis , grade and stage of tumor was carried out after reviewing all slides before proceeding to the immunohistochemical approach . tissue sections with a thickness of 5- were taken from the formalin - fixed , paraffin embedded blocks for immunohistochemistry . labeled streptavidin - biotin ( lsab ) method was employed for immunohistochemical detection of her-2/neu using hercep test kit ( k5204 dako co. ) . the intensity of her-2/neu cell membrane stain was classified into score 0 ( negative ; no stain is observed or faint membrane staining presents in less than 10% of tumor cells ) , score 1 + ( negative ; a faint / barely perceptible membrane staining was detected in more than 10% of tumor cells ; these cells exhibit incomplete membrane staining ) , score 2 + ( weakly positive ; a weak to moderate complete membrane staining was observed in more than 10% of tumor cells ) and score 3 + ( strongly positive ; a strong complete membrane staining was observed in more than 10% of tumor cells ) . all biopsies were classified according to the modified bloom richardson grading system into three grades : grade i , grade ii and grade iii . the results were statistically evaluated with a chi - squared test ( at a significant level of < 0.05 ) and correlation - regression analysis ( at significant level of r = 0.3 ) using ssps software . the study received ethical approval from the research ethics committee of the middle euphrates centre for cancer research and followed the tenets of the declaration of helsinki . her-2/neu immuno - expression was positive in 67.8% of breast cancer cases and negative in all sections of the normal breast tissue and benign breast lesions ( fibroadenomas ) with significant differences among these groups ( p < 0.05 ) [ table 1 ] . her-2/neu overexpression was detected in 73.2% of invasive ductal carcinoma ( idc ) cases and in 12.5% of invasive lobular carcinoma . her-2/neu(c - erbb-2 ) overexpression and histological characteristics figures in parentheses are in percentage overexpression of her-2/neu was detected in 65.6% of those with pure invasive ductal carcinomas ; all of them were of a nonspecific type . a 94.4% of invasive ductal carcinoma with an in situ comedo component ( dcis ) and 100% of invasive ductal carcinoma with overlying paget 's disease were her-2/neu positive with a significant difference ( p < 0.05 ) in comparison with pure invasive ductal carcinomas [ table 1 ] . there was a significant difference between the intensity of her-2/neu overexpression and the histological type of breast cancer ( p < 0.05 ) [ table 2 ] . her-2/neu overexpression was detected in 70.6% of grade ii and 69.0% of grade iii breast cancer samples , while none of grade i showed her-2/neu overexpression [ table 1 ] . there was no significant difference in her-2/neu overexpression between grade ii and grade iii of breast cancer ( p > 0.05 ) , but in comparison with grade i a significant difference was noticed ( p < 0.05 ) . her-2/neu overexpression was highly correlated with grade of tumor ( r = 0.96 ) , indicating that her-2/neu - positive breast cancers are biologically aggressive . no statistically significant difference in the correlation between the intensity of her-2/neu immunostaining and histological grade was found ( p > 0.05 ) [ table 2 ] . the intensity of her-2/neu(c - erbb-2 ) overexpression and histological characteristics in addition , a higher detection rate of her-2/neu overexpression was noticed in recurrent breast cancer than primary lesions ( 86.2% and 59.1% , respectively ) with a significant difference ( p < 0.05 ) [ table 1 ] . no statistically significant differences between her-2/neu overexpression and tumor size , tumor stage , lymph node involvement , and age of breast cancer patients were found ( p > 0.05 ) [ table 1 ] . depleted uranium levels were estimated to be around 320800 tons in the aftermath of the first gulf war in 1991 with further comparable levels occurring in 2003 . since the targets were always in heavily populated areas in the middle and south of iraq , the extent of exposure on individuals was extensive but has been very hard to document accurately . this study showed that her-2/neu overexpression was completely absent in both normal breast tissue and benign lesion ( fibroadenomas ) sections . the percentage of her-2/neu overexpression in malignant breast lesions is disputed in our study as it ranges from 12.5% to 100% , but the finding is that her-2/neu overexpression appears to be a biomarker for malignant breast tissue and does not play any role in the benign ( fibroadenomas ) breast lesions . this observation confirms the results of many previous studies that reached the same conclusion.[1112 ] indeed , our study reported her-2/neu overexpression in 67.8% out of 90 breast cancer cases [ figure 1a ] . furthermore , most of the ductal carcinoma cases were purely invasive ductal carcinomas of nonspecific type ( 74.4% ) that showed significant her-2/neu overexpression ( 65.6% ) . this provides more evidence of the hypothesis that aggressive tumors seem to show significant her-2/neu overexpression and demonstrates the association between the nature of the biological expression of her-2/neu by the tumor and its degree of malignancy since it has been argued that nonspecific type ductal carcinomas are the most aggressive variants of breast cancer . the current study also demonstrated that 12.5% of lobular carcinoma cases exhibited her-2/neu expression [ figure 1d ] . ( a ) invasive ductal carcinoma , ( ) showing strong complete cytoplasmic membrane staining of score 3 + ( 40 ) . ( b ) insitu ductal carcinoma of comedo type , ( ) showing strong complete cytoplasmic membrane staining of score 3+positive ( 10 ) . ( c ) paget 's disease , ( ) showing strong complete cytoplasmic membrane staining of score 3 + ( 40 ) . ( d ) invasive lobular carcinoma , pleomorphic type , ( ) showing moderate complete membrane staining of score 2 + ( 40 ) from the above results , a significant difference in her-2/neu overexpression was observed between invasive ductal and lobular carcinoma ( p < 0.05 ) . this high discrepancy in her-2/neu overexpression between invasive ductal and lobular carcinoma suggests that invasive lobular carcinomas are both morphologically and biologically different from invasive ductal carcinomas . however , further studies are required to provide a more definitive answer . all 18 cases of idc with comedo - type in situ component showed her-2/neu overexpression [ figure 1b ] . the finding that her-2/neu expression is seen more frequently in dcis than in invasive carcinoma implicates that her-2/neu signaling pathway is playing a critical role in the early stages of breast tumorogenesis . overexpression was seen in all cases of both the invasive ductal carcinoma and paget 's disease components , when both components were seen simultaneously [ figure 1c ] . her-2/neu overexpression of low intensity ( score 1 + , considered as negative ) was observed more frequently in ilc ( 50% ) , while weak to moderate intensity of her-2/neu overexpression ( score 2 + ) occurred more frequently in invasive ductal carcinoma ( idc ) + ductal carcinoma with an in situ comedo component ( dcis ) ( 55.5% ) . high intensity of her-2/neu overexpression ( score 3 + , considered as strong positive ) was reported more frequently in paget 's disease ( 100% ) . these results suggest that the intensity of her-2/neu overexpression is well correlated with histopathological type . previous studies have shown faint or absent her-2/neu overexpression in lobular carcinoma and a strong positive score ( score + 3 ) in all cases of paget 's disease . the findings of this study show that a high incidence of samples of idc with in - situ component ( 55.5% ) were moderately stained ( score + 2 ) . this compares with the findings of lan et al . , who examined the prevalence of c - erbb-2 gene amplification in moderately stained ( score + 2 ) taiwanese specimens using ihc and found a higher prevalence of her-2 overexpression ( 44.4% ) than that found in similar studies from western countries ( 13%-23% ) . this study found no significant association between her-2/neu overexpression and age of the patients [ table 1 ] , which concurs with a previous study . a high proportion of her-2/neu overexpression was seen in moderately and poorly differentiated cases of breast cancer , while a lower proportion of her-2/neu expression was reported in cases of well differentiated breast cancer . her-2/neu expression appears to be well correlated with tumor grade ( r = 0.96 ) . immunohistochemical analysis showed her-2/neu overexpression to be higher in recurrent than in primary cases of tumor . these results reflect the significant role of her-2/neu overexpression in increasing the risk of local recurrence . it has been well documented that positive her/2 breast cancer patients have good prognosis as they respond well to the blocking effect of these receptors by anti - her/2 receptor antibody using monoclonal ( herceptin ) drug that plays a role in the regression of the tumor size and prevention of recurrence . the driving factor for the increased expression of her-2/neu to the level of 68% compared to general expression of 30% might be due to some underlying genetic factors , although other environmental factors can not be excluded . further investigations are needed to ascertain the extent of the effect of depleted uranium and the types of mutations noted in the overexpressed oncogene before any final conclusions can be reached despite the three - fold increase of all types of breast cancer in iraq due to long - term exposure to depleted uranium , this heightened exposure does not appear to be correlated with her-2/neu overexpression . further longitudinal studies and the use of techniques such as microarrays are required to investigate whether any effect of depleted uranium does manifest in the pathology of breast cancer . infantis resulted from a general increase in different biotypes or a successful spread of 1 clone . infantis identified in israel during 20072009 ( 21 human sources , 28 poultry sources , and 22 food sources ) and 16 historical strains isolated during 19702005 ( 12 human sources , 2 poultry sources , and 2 food sources ) were subjected to pulsed - field gel electrophoresis ( pfge ) . macrorestriction with the xbai enzyme discriminated the isolates into 23 distinct profiles ( pulsotypes ) , designated i1i23 . although the historical isolates showed high diversity in their pfge patterns , most ( 58/71 , 82% ) recent ( 20072009 ) isolates were homogeneous and showed an indistinguishable pfge profile ( pulsotype i1 ) , which was not found among the historical isolates ( figure 2 ; table a1 ) . these results indicate that most of the emerging isolates belong to 1 genetic clone that probably started to spread in israel sometime during 20052007 . furthermore , comparison of the i1 pulsotype with other pfge profiles through pulsenet ( www.cdc.gov/pulsenet/ ) and pulsenet europe ( www.pulsenetinternational.org/networks/europe.asp ) indicated a pattern not reported elsewhere , suggesting the emerging clone is endemic to israel . pulsed - field gel electrophoresis ( pfge ) patterns of salmonella enterica serovar infantis isolates from clinical , food , and poultry sources isolated in israel , 19702009 , showing a high degree of clonality . macrodigestion performed using xbai restriction enzyme and genetic similarity ( in % ) was based on dice coefficients . pfge was conducted according to the standardized salmonella protocol centers for disease prevention and control pulsenet as described ( 4 ) by using s. enterica ser . a complete list is provided in table a1 . to further characterize the isolates , we performed susceptibility tests to 16 antimicrobial compounds . overall , resistance to 11 antimicrobial agents was detected ( table ; table a1 ) . first , although 6/16 ( 38% ) of the historical strains were sensitive to all tested antimicrobial agents and 5/16 ( 31% ) were resistant to only 1 ( nitrofurantoin ) , none of the 20072009 isolates were sensitive to all of the tested antimicrobial agents . most ( 68/71 , 96% ) of the recent isolates were resistant to > 3 antimicrobial agents , which suggests a process of resistance acquisition over time . second , whereas isolates from 19702005 did not share any obvious resistance pattern , most ( 66/71 , 93% ) of the 20072009 strains showed a combined resistance pattern to nalidixic acid , nitrofurantoin , and tetracycline with or without additional resistance to trimethoprim / sulfamethoxazole ( table ) . the convergence of the recent serovar infantis clones to a dominant resistance pattern is consistent with their common pfge profile and shows that they share high similarity on phenotypic and genotypic levels . * pfge , pulsed - field gel electrophoresis ; i1 , emerging pfge pattern ; d , different from the emerging pattern . next , we characterized the molecular mechanisms responsible for the common antimicrobial drug resistance phenotype . in bacteria , an efficient means of acquisition and dissemination of resistance genes is through mobile genetic elements such as plasmids , transposons , or integrons ( 5 ) . plasmid analysis for 15 emerging ( 20072009 ) and 7 historical ( 19702005 ) randomly selected isolates demonstrated that all possessed 1 large plasmid of 100 kb . to identify antimicrobial drug resistance genes that are possibly encoded on this plasmid , mating experiments were conducted with a plasmid - free , rifampin - resistant escherichia coli j53 strain and recent s. enterica ser . conjugation experiments showed the obtained e. coli transconjugants received the large ( 100-kb ) plasmid and acquired the tetracycline resistance phenotype but remained susceptible to nalidixic acid and nitrofurantoin . molecular analysis by pcr showed the teta gene encoded within the tn1721 transposon in 6 of 6 randomly selected emerging isolates but in only 1 of 5 older historical strains . we examined class 1 integrons using pcr primers designed to amplify the variable region of class 1 integrons . sulfamethoxazole followed by the orfc gene of unknown function . in contrast , 3/5 historical isolates did not possess any integron , and 2/5 contained a disparate integron with a variable region of 1.3 kb . sequencing analysis indicated a different cassette encoded by the aminoglycoside adenyltransferase aada1 gene conferring resistance to spectinomycin and streptomycin . resistance to quinolones is often associated with point mutations in the quinolone - resistance determining region of the gyra gene ( 6 ) . to examine this possibility , we determined the gyra sequence from 6 recent naladixic acid resistant and 4 naladixic acid sensitive isolates . all resistant clones showed the same nucleotide substitution from guanine to thymine at position 259 ( g259 t ) in the gyra gene , resulting in the exchange of asparagine in position 87 to tyrosine ( asp87tyr ) in the quinolone resistance determining region domain . no mutations were found in the gyra sequence of the naladixic acid sensitive isolates , suggesting that the asp87tyr point mutation is responsible for the observed naladixic acid resistance phenotype . it is likely that environmental selective pressure caused by use of antimicrobial drugs has led to the distribution of appropriate resistant genes . nitrofurans and sulfonamides , for example , have been widely used to treat infections and promote growth of livestock ( 7 ) . because the emerging clone was dominant in all levels of the food chain , including broiler chickens , it is possible that the emerging clone was originally introduced from a poultry source . recent studies from other countries identified healthy poultry as a potential reservoir of s. enterica ser . infantis isolates from different sources and regions in israel showed high homogeneity of emerging isolates that differ genetically and phenotypically from previously isolated strains . we showed that the emerging clone is multidrug resistant and is characterized by a large conjugative plasmid harboring the tn1721 transposone and teta gene , which provides reduced susceptibility to tetracyclines . additional characteristics include a class 1 integron containing the dfra1 cassette , a gyra mutation that mediates nalidixic acid resistance and furthers resistance to nitrofurantoin . our results suggest the recent emergence of serovar infantis is an outcome of a clonal expansion and establishment of a specific biotype that took place during a relatively short period .

backgroundthis study aimed to estimate the rate of her-2/neu ( c - erbb2 ) immunohistochemical overexpression in different histological types of breast cancer found in the middle euphrates region of iraq , a region that was exposed to high levels of depleted uranium . her-2/neu ( c - erbb2 ) overexpression was correlated with common clinicopathological parameters such as age , grade , stage , tumor size and lymph node involvement to determine if any particular biomarker for exposure to depleted uranium could be found in the tumor samples from this region.materials and methodsthe present investigation was performed over a period starting from september 2007 to june 2008 . formalin - fixed , paraffin - embedded blocks from 90 patients with breast cancer were included in this study . a group of 25 patients with benign breast lesions ( fibroadenoma ) was included as a comparative group , and 20 breast tissue sections were used as controls . labeled streptavidin - biotin ( lsab ) complex method was employed for immunohistochemical detection of her-2/neu.resultsher-2/neu immuno - expression was positive in 67.8% of breast cancer , while it was negative in all benign breast lesions ( fibroadenoma ) ( p < 0.05 ) . her-2/neu immunostaining was significantly associated with histological type and recurrence of breast cancer ( p < 0.05 ) . it was positively correlated with tumor grade , but this finding was not significant ( p > 0.05).conclusionbased upon the findings of this study , it can be concluded that her-2/neu overexpression plays an important role in the pathogenesis of breast cancer and is associated with a worse prognosis . the findings indicate that in regions exposed to high levels of depleted uranium , her-2/neu overexpression is high , but its correlation with age , grade , stage , tumor size , and lymph node involvement is similar to studies that have been conducted on populations not exposed to depleted uranium . to determine whether rapid emergence of salmonella enterica serovar infantis in israel resulted from an increase in different biotypes or spread of 1 clone , we characterized 87 serovar infantis isolates on the genotypic and phenotypic levels . the emerging strain comprised 1 genetic clone with a distinct pulsed - field gel electrophoresis profile and a common antimicrobial drug resistance pattern .

cancer vaccine targeting hepatocellular carcinoma ( hcc ) tumor antigens have been tested in clinical trials . however , cancer vaccines using tumor - antigen - derived peptides have not demonstrated adequate antitumor efficacy in clinical trials for advanced hcc . glypican-3 ( gpc3 ) , a carcinoembryonic antigen , is an ideal target for immunotherapy against hcc because it is overexpressed specifically in hcc ( 7281% ) and correlates with a poor prognosis . gpc3 forms a complex with wnt molecules and promotes the growth of hcc by stimulating canonical wnt signaling . we identified hla - a*24:02-restricted gpc3298306 ( eyilsleel ) and hla - a*02:01-restricted gpc3144 - 152 ( fvgefftdv ) peptides , both of which induce gpc3-reactive cytotoxic t - lymphocytes ( ctls ) without inducing autoimmunity . we recently reported the safety , immunological , and clinical responses of a gpc3-derived peptide vaccine in a phase i clinical trial of patients with advanced hcc . the results of that trial showed that gpc3 peptide - specific ctls increased in peripheral blood , and that many cd8-positive t cells infiltrated the tumors in some patients , demonstrating a correlation between the ctl response and overall survival following gpc3 peptide vaccination . based on these results , we conducted a trial in patients with advanced hcc to assess the clinical outcome and whether tumor - infiltrating lymphocytes with an antitumor effect increased . in all cases , liver biopsies were performed before and after gpc3 peptide vaccination according to the protocol . this trial was approved by the ethics committee of the national cancer center and registered with the university hospital medical information network clinical trials registry ( umin - ctr number 000005093 ) . the patient described herein was the first case examined pathologically using autopsy specimens . here , we present the clinical course and pathological study , including an autopsy , of a patient with advanced hcc who revealed remarkable tumor lysis immediately after the second vaccination in an ongoing clinical trial of a gpc3 peptide vaccine . a 62-year - old male had a history of asymptomatic chronic hepatitis c. in september 2009 , he was diagnosed with hcc . abdominal ct ( ct ) scans showed four lesions in the liver , and the patient was treated four times with hepatic artery chemoembolization . in december 2010 , ct scans revealed a new lesion indicative of a tumor thrombus extending into the inferior vena cava . . however , the sorafenib treatment was discontinued in january 2011 due to progressive multiple intrahepatic tumors . as no established therapeutic regimens exist for this condition , he was offered participation in a clinical trial of a gpc3 peptide vaccine for advanced hcc . the patient had a performance status of 0 , and child - pugh class b disease . the patient did not have active hbv infection or rapidly progressive tumor thrombus before enrollment , met the eligibility criteria , and was enrolled after providing informed consent . early - phase contrast - enhanced ct before treatment showed a maximum 68 51-mm tumor with multiple intrahepatic tumors and a 44 30-mm tumor invading the right atrium ( fig . pretreatment tumor markers were as follows : fetoprotein ( afp ) , 852 ng / ml and des - gamma - carboxy prothrombin ( dcp ) , 1346 mau / ml . a liver biopsy was performed 1 week prior to gpc3 peptide vaccination according to the protocol . in april 2011 , 3 mg of hla - a2-restricted gpc3144 - 152 peptide ( fvgefftdv ) ( american peptide co. ) emulsified with incomplete freund s adjuvant ( montanide isa-51vg ; seppic ) was injected intradermally as the vaccine following good manufacturing practice guidelines . the patient had a low - grade fever on day 6 following the first vaccination , and inflammatory and hepatic parameters were elevated on day 12 ( fig . therefore , he received the second vaccination on day 26 after the first vaccination . on day 9 after the second vaccination , the patient was admitted to our hospital with a high fever and general fatigue . on admission , the patient s c - reactive protein ( crp ) level ( 10.76 mg / dl ) and laboratory hepatic parameters were elevated . one day after hospitalization , aspartate aminotransferase and alanine aminotransferase and levels were elevated to 1,580 as seen by early - phase contrast - enhanced ct scan , most tumors in the liver were not contrast enhanced . in contrast , the size of the tumor thrombus in the right atrium increased to a maximum of 83 50 mm ( fig . levels of the tumor markers afp and dcp decreased temporarily to 634 ng / ml and 777 mau / ml , respectively . he was infused with a liver - supporting agent ( monoammonium glycyrrhizinate , glycine , and l - cysteine hydrochloride hydrate ) . we did not perform a liver biopsy when the hepatic parameters were elevated because they improved promptly . protocol treatment was discontinued due to progressive disease and he died 30 days after the second vaccination . based on the clinical course , we could not rule out the possibility that his condition had worsened as a result of the vaccine . therefore , an autopsy was performed to determine the main cause of death and the elevated hepatic parameters , and to evaluate the anti - tumor effect of vaccination figure 1 . ( a ) contrast - enhanced ct scan before vaccination shows a 68 51-mm tumor with multiple intrahepatic tumors ( arrow ) and a 44 30-mm tumor invading the right atrium ( arrowhead ) . ( b ) contrast - enhanced ct after the second vaccination showing multiple low - density areas in the liver , indicating extensive tumor necrosis ( arrow ) . by contrast , a tumor thrombus in the right atrium increased to a 83 50-mm tumor ( arrowhead ) . approximately 1 week after the first vaccination , the patient began reporting general fatigue and showed intermittent fever . inflammatory and hepatic parameters were elevated ( crp : pink line , ast : red line , t - bil : green line ) . the abnormal laboratory parameters improved after observation . on day 9 after the second vaccination , the patient was admitted to our hospital as an emergency due to fever and general fatigue , which were similar to his previous symptoms . inflammatory and hepatic parameters improved 1 week after hospitalization . however , his status gradually worsened , and he died on day 30 after the second vaccination . ex vivo ifn- enzyme - linked immunospot ( elispot ) assays against gpc3 in 5 10 peripheral blood mononuclear cells ( pbmcs ) were performed before and after vaccination . the spot number indicates the number of gpc3 peptide - specific cytotoxic t - lymphocytes ( ctls ) . the number of interferon ( ifn)- positive spots increased from 0 to 84 after the second vaccination . a 62-year - old male had a history of asymptomatic chronic hepatitis c. in september 2009 , he was diagnosed with hcc . abdominal ct ( ct ) scans showed four lesions in the liver , and the patient was treated four times with hepatic artery chemoembolization . in december 2010 , ct scans revealed a new lesion indicative of a tumor thrombus extending into the inferior vena cava . . however , the sorafenib treatment was discontinued in january 2011 due to progressive multiple intrahepatic tumors . as no established therapeutic regimens exist for this condition , he was offered participation in a clinical trial of a gpc3 peptide vaccine for advanced hcc . the patient had a performance status of 0 , and child - pugh class b disease . the patient did not have active hbv infection or rapidly progressive tumor thrombus before enrollment , met the eligibility criteria , and was enrolled after providing informed consent . early - phase contrast - enhanced ct before treatment showed a maximum 68 51-mm tumor with multiple intrahepatic tumors and a 44 30-mm tumor invading the right atrium ( fig . pretreatment tumor markers were as follows : fetoprotein ( afp ) , 852 ng / ml and des - gamma - carboxy prothrombin ( dcp ) , 1346 mau / ml . a liver biopsy was performed 1 week prior to gpc3 peptide vaccination according to the protocol . in april 2011 , 3 mg of hla - a2-restricted gpc3144 - 152 peptide ( fvgefftdv ) ( american peptide co. ) emulsified with incomplete freund s adjuvant ( montanide isa-51vg ; seppic ) was injected intradermally as the vaccine following good manufacturing practice guidelines . the patient had a low - grade fever on day 6 following the first vaccination , and inflammatory and hepatic parameters were elevated on day 12 ( fig . therefore , he received the second vaccination on day 26 after the first vaccination . on day 9 after the second vaccination , the patient was admitted to our hospital with a high fever and general fatigue . on admission , the patient s c - reactive protein ( crp ) level ( 10.76 mg / dl ) and laboratory hepatic parameters were elevated . one day after hospitalization , aspartate aminotransferase and alanine aminotransferase and levels were elevated to 1,580 iu / l and 1,112 as seen by early - phase contrast - enhanced ct scan , most tumors in the liver were not contrast enhanced . in contrast , the size of the tumor thrombus in the right atrium increased to a maximum of 83 50 mm ( fig . levels of the tumor markers afp and dcp decreased temporarily to 634 ng / ml and 777 mau / ml , respectively . he was infused with a liver - supporting agent ( monoammonium glycyrrhizinate , glycine , and l - cysteine hydrochloride hydrate ) . we did not perform a liver biopsy when the hepatic parameters were elevated because they improved promptly . protocol treatment was discontinued due to progressive disease and he died 30 days after the second vaccination . based on the clinical course , we could not rule out the possibility that his condition had worsened as a result of the vaccine . therefore , an autopsy was performed to determine the main cause of death and the elevated hepatic parameters , and to evaluate the anti - tumor effect of vaccination figure 1 . ( a ) contrast - enhanced ct scan before vaccination shows a 68 51-mm tumor with multiple intrahepatic tumors ( arrow ) and a 44 30-mm tumor invading the right atrium ( arrowhead ) . ( b ) contrast - enhanced ct after the second vaccination showing multiple low - density areas in the liver , indicating extensive tumor necrosis ( arrow ) . by contrast , a tumor thrombus in the right atrium increased to a 83 50-mm tumor ( arrowhead ) . approximately 1 week after the first vaccination , the patient began reporting general fatigue and showed intermittent fever . inflammatory and hepatic parameters were elevated ( crp : pink line , ast : red line , t - bil : green line ) . the abnormal laboratory parameters improved after observation . on day 9 after the second vaccination , the patient was admitted to our hospital as an emergency due to fever and general fatigue , which were similar to his previous symptoms . however , his status gradually worsened , and he died on day 30 after the second vaccination . ex vivo ifn- enzyme - linked immunospot ( elispot ) assays against gpc3 in 5 10 peripheral blood mononuclear cells ( pbmcs ) were performed before and after vaccination . the spot number indicates the number of gpc3 peptide - specific cytotoxic t - lymphocytes ( ctls ) . the number of interferon ( ifn)- positive spots increased from 0 to 84 after the second vaccination . generally , ctls specific for tumor antigens can not be detected directly ex vivo ; they can be detected only after expansion by repeated in vitro stimulation with the antigenic peptide in conjunction with appropriate antigen - presenting cells . this is attributed to the sensitivity of the assay and the low frequency of tumor - antigen - specific ctls . gpc3 peptide - specific ctls in pbmcs , which can be detected directly ex vivo without in vitro stimulation , can provide strong immunological evidence . the number of gpc3 peptide - specific ctls increased from 0 to 84 in 5 10 pbmcs after the second vaccination ( fig . this result led us to anticipate a good clinical response because the increased number of ctls and the specific ctl number correlated with the clinical response in a previous trial of the gpc3 peptide vaccine . immunohistochemical staining showed expression of gpc3 and hla class i in the cytoplasm and membranes of the carcinoma cells and a few cd8-positive t cells in the carcinoma tissue before vaccination ( fig . ( a ) pathological findings of liver biopsy specimens before vaccination . a microscopy image of a hematoxylin and eosin ( h&e)-stained section shows well - differentiated hepatocellular carcinoma ( hcc ) . immunohistochemical staining for gpc3 and hla class i showed positivity in the cytoplasm and membranes of carcinoma cells , respectively . ( b ) macroscopic findings of the liver and heart before formalin fixation at the time of autopsy . ( a ) microscopic images of h&e - stained sections showing central necrosis of carcinoma tissue , whereas a cirrhotic nodule adjacent to the carcinoma tissue was not necrotic . ( b ) magnified image of the area enclosed within the white box in ( a ) showing that cancer cells exhibited a morphology ( left ) different from that of cirrhotic cells ( right ) . ( c ) cd8-positive t cells ( brown ) infiltrated the carcinoma cells accompanied by necrosis . in contrast , no infiltration of cd8-positive t cells was detected within the cirrhotic nodule . ( d ) magnified image of the area enclosed within the red box in ( a ) showing necrosis and viable carcinoma cells . ( e ) positive immunohistochemical gpc3 staining was observed in only the cytoplasm of carcinoma cells . ( f ) cd8-positive t cells infiltrated the necrotic area and carcinoma tissue . a general autopsy ( with the exception of the brain ) was performed 2 h following death . macroscopic findings of the liver revealed multiple macro - nodular lesions with central necrosis mainly in the right lobe ( fig . as the tumor occupied most of the right atrium , the main cause of death was circulatory failure due to progressive tumor thrombus ( fig . 2b , right ) . a histological examination showed central necrosis in most of the tumor in the right lobe , and viable carcinoma cells remained around the necrotic tissue , whereas a cirrhotic nodule adjacent to the carcinoma tissue was not necrotic ( figs . 2c and 3a ) . there was infiltration of cd8-positive t cells ( brown ) in the residual carcinoma , but not within the cirrhotic area ( figs . 2c and 3a ) . we did not detect degeneration or necrosis of the hepatocytes in the non - tumor liver parenchyma of the left lobe . these findings suggest that the elevated hepatic parameters in our patient were due to an antitumor effect . we diagnosed that the cause of death was unlikely to be related to vaccine - induced liver injury . we focused on the necrotic area around the cirrhotic nodules , in which cd68-positive macrophages ( brown ) aggregated ( fig . cd8-positive t cells also infiltrated the marginal zone between the necrotic area and noncancerous cirrhotic nodule , suggesting that carcinoma cells were attacked by cd8-positive t cells , which may have resulted in necrosis ( fig . the histology of the tumor thrombus in the right atrium was similar to that of the intrahepatic tumor . however , viable tumor cells remained in half of the tumor thrombus and little infiltration of cd8-positive t cells was detected ( data not shown ) . cd8-positive t cells ( brown ) infiltrated only the carcinoma area , accompanied by necrosis . ( c ) cd68-positive macrophages ( brown ) aggregated in the necrotic area around the cirrhotic nodule . ( d ) cd8-positive t cells ( brown ) infiltrated the necrotic area but not the cirrhotic nodule . based on the image in ( d ) , most of the lymphocytes were cd8-positive t cells . generally , ctls specific for tumor antigens can not be detected directly ex vivo ; they can be detected only after expansion by repeated in vitro stimulation with the antigenic peptide in conjunction with appropriate antigen - presenting cells . this is attributed to the sensitivity of the assay and the low frequency of tumor - antigen - specific ctls . gpc3 peptide - specific ctls in pbmcs , which can be detected directly ex vivo without in vitro stimulation , can provide strong immunological evidence . the number of gpc3 peptide - specific ctls increased from 0 to 84 in 5 10 pbmcs after the second vaccination ( fig . this result led us to anticipate a good clinical response because the increased number of ctls and the specific ctl number correlated with the clinical response in a previous trial of the gpc3 peptide vaccine . immunohistochemical staining showed expression of gpc3 and hla class i in the cytoplasm and membranes of the carcinoma cells and a few cd8-positive t cells in the carcinoma tissue before vaccination ( fig . ( a ) pathological findings of liver biopsy specimens before vaccination . a microscopy image of a hematoxylin and eosin ( h&e)-stained section shows well - differentiated hepatocellular carcinoma ( hcc ) . immunohistochemical staining for gpc3 and hla class i showed positivity in the cytoplasm and membranes of carcinoma cells , respectively . ( b ) macroscopic findings of the liver and heart before formalin fixation at the time of autopsy . ( a ) microscopic images of h&e - stained sections showing central necrosis of carcinoma tissue , whereas a cirrhotic nodule adjacent to the carcinoma tissue was not necrotic . ( b ) magnified image of the area enclosed within the white box in ( a ) showing that cancer cells exhibited a morphology ( left ) different from that of cirrhotic cells ( right ) . ( c ) cd8-positive t cells ( brown ) infiltrated the carcinoma cells accompanied by necrosis . in contrast , no infiltration of cd8-positive t cells was detected within the cirrhotic nodule . ( d ) magnified image of the area enclosed within the red box in ( a ) showing necrosis and viable carcinoma cells . ( e ) positive immunohistochemical gpc3 staining was observed in only the cytoplasm of carcinoma cells . ( f ) cd8-positive t cells infiltrated the necrotic area and carcinoma tissue . a general autopsy ( with the exception of the brain ) was performed 2 h following death . macroscopic findings of the liver revealed multiple macro - nodular lesions with central necrosis mainly in the right lobe ( fig . as the tumor occupied most of the right atrium , the main cause of death was circulatory failure due to progressive tumor thrombus ( fig . 2b , right ) . a histological examination showed central necrosis in most of the tumor in the right lobe , and viable carcinoma cells remained around the necrotic tissue , whereas a cirrhotic nodule adjacent to the carcinoma tissue was not necrotic ( figs . 2c and 3a ) . there was infiltration of cd8-positive t cells ( brown ) in the residual carcinoma , but not within the cirrhotic area ( figs . 2c and 3a ) . we did not detect degeneration or necrosis of the hepatocytes in the non - tumor liver parenchyma of the left lobe . these findings suggest that the elevated hepatic parameters in our patient were due to an antitumor effect . we diagnosed that the cause of death was unlikely to be related to vaccine - induced liver injury . we focused on the necrotic area around the cirrhotic nodules , in which cd68-positive macrophages ( brown ) aggregated ( fig . cd8-positive t cells also infiltrated the marginal zone between the necrotic area and noncancerous cirrhotic nodule , suggesting that carcinoma cells were attacked by cd8-positive t cells , which may have resulted in necrosis ( fig . the histology of the tumor thrombus in the right atrium was similar to that of the intrahepatic tumor . however , viable tumor cells remained in half of the tumor thrombus and little infiltration of cd8-positive t cells was detected ( data not shown ) . cd8-positive t cells ( brown ) infiltrated only the carcinoma area , accompanied by necrosis . ( c ) cd68-positive macrophages ( brown ) aggregated in the necrotic area around the cirrhotic nodule . ( d ) cd8-positive t cells ( brown ) infiltrated the necrotic area but not the cirrhotic nodule . based on the image in ( d ) , most of the lymphocytes were cd8-positive t cells . to date , the time to ctl induction and subsequent tumor response has been prolonged in cancer vaccine trials . by contrast , no discrepancy regarding the time between ctl induction and tumor response was observed in our phase i trial of a gpc3 peptide vaccine . in this case , central necrosis of each intra - hepatic tumor was observed at the time of a strong immunological response against the gpc3 peptide , immediately after the second vaccination . a biopsy may be necessary to rule out vaccine - induced liver injury when the hepatic parameters are elevated . however , the clinical course and autopsy results suggested that the elevated hepatic parameters in our patient were due to an antitumor effect . therefore , a positive radiographic response following vaccination , suggesting tumor necrosis , could be evaluated as a treatment response . necrosis was found in the center of each tumor ; therefore , the central necrosis caused by ischemia , in addition to cd8-positive t cells attacking tumor cells , may have led to tumor necrosis . three findings support the hypothesis that tumor necrosis was caused by cd8-positive t cells , as follows : ( 1 ) the necrotic changes determined by ct after vaccination , accompanied by clinical laboratory data ; this was consistent with an immune response , although no tumor necrosis was evident on the ct before vaccination ; ( 2 ) no necrosis was evident in the left lobe ( no tumors ) of the autopsy liver specimen , but it was present in the right liver lobe ( tumors present ) ; and ( 3 ) cd8-positive t cells infiltrated residual viable tumor cells . the analyses used in this study may contribute to identifying the pathological state after vaccination . we detected infiltration of cd8-positive t cells into the hepatic tumors , but little infiltration of cd8-positive t cells into the tumor thrombus . this discrepancy may have been caused by the heterogeneity associated with immune - escape mechanisms in tumor cells . this case report of central necrosis in a patient with hcc might be regarded as spontaneous regression correlated with circulatory failure due to a massive tumor embolism . it was not known whether the tumor necrosis was induced by ctls , ischemia , or other factors . however , the infiltration of cd8-positive t cells into tumor cells supports immune - related necrosis . the rate of spontaneous partial regression among patients with hcc is 0.406% compared with the control arm of a randomized clinical trial . in contrast , three of 33 patients who received gpc3 peptide vaccination in the phase i trial had suspicious tumor necrosis on ct scans . in one report , massive infiltration of cd8-positive t cells in the remaining liver tumor and tumor necrosis were identified by histological examination of a biopsy specimen after vaccination . indeed , on - going clinical trials of the gpc3 peptide vaccine will provide additional information and further demonstrate the antitumor effect . histological results at the estimated time of a strong gpc3-specific ctl response suggest that gpc3 peptide vaccination may be a promising approach to treat hcc . an ex vivo ifn- enzyme - linked immunospot ( elispot ) assay was performed to evaluate the antigen - specific ctl response , as described previously . briefly , peripheral blood ( 30 ml ) was obtained from the patient before the first vaccination and 2 weeks after each vaccination and centrifuged on a ficoll non - cultured peripheral blood mononuclear cells ( pbmcs ) ( 5 10/well ) were added to plates in the presence of 10 g / ml peptide antigens and incubated for 20 h. the gpc3 antigen used was the hla - a2-restricted gpc3144152(fvgefftdv ) peptide . pbmcs with the hla - a2-restricted hiv1927 ( tlnawvkvv ) peptide ( proimmune ) were used as negative controls . immunohistochemical staining with monoclonal antibodies against gpc3 ( clone , 1g12 ; biomosaics ) , hla class i ( clone , emr8/5 ; hokudo ) , cd8 ( clone , 1a5 ; novocastra ) , cd45 ( cloned 2b11 and pd7/26 ; ventana ) , and cd68 ( clone , kp-1 ; ventana ) was performed according to the manufacturer s protocol . an ex vivo ifn- enzyme - linked immunospot ( elispot ) assay was performed to evaluate the antigen - specific ctl response , as described previously . briefly , peripheral blood ( 30 ml ) was obtained from the patient before the first vaccination and 2 weeks after each vaccination and centrifuged on a ficoll non - cultured peripheral blood mononuclear cells ( pbmcs ) ( 5 10/well ) were added to plates in the presence of 10 g / ml peptide antigens and incubated for 20 h. the gpc3 antigen used was the hla - a2-restricted gpc3144152(fvgefftdv ) peptide . pbmcs with the hla - a2-restricted hiv1927 ( tlnawvkvv ) peptide ( proimmune ) were used as negative controls . immunohistochemical staining with monoclonal antibodies against gpc3 ( clone , 1g12 ; biomosaics ) , hla class i ( clone , emr8/5 ; hokudo ) , cd8 ( clone , 1a5 ; novocastra ) , cd45 ( cloned 2b11 and pd7/26 ; ventana ) , and cd68 ( clone , kp-1 ; ventana ) was performed according to the manufacturer s protocol . research into the use of cell therapy for cardiac regeneration has undergone explosive growth over the prior two decades in both pre - clinical models of infarct healing and in numerous clinical trials . while early studies sought to replace damaged myocardium with implantation of autologous satellite cells ( for a fascinating review of early cardiac regeneration research see yoon et al . ) , a variety of stem cells with differing origins and levels of pluripotency have been proposed and tested in recent years [ 2 , 3 ] . overwhelmingly , most studies have demonstrated a transient improvement in global cardiac function following cell implantation or homing . however , the fraction of cells that survive to later time points is typically below 1 % . the processes underlying the high rates of death amongst injected cells , and potential mechanisms by which to promote increased cell survival and differentiation remain poorly understood . a major barrier to elucidating these processes is the inability to serially examine cell fate decisions following implantation in a non - invasive and in vivo manner [ 57 ] . magnetic resonance imaging ( mri ) has become a reference standard modality for non - invasive imaging of soft tissue with superior contrast . when applied to imaging of the heart , cardiac mri ( cmr ) is emerging as a cutting edge modality capable of enabling multi - scale characterization of the heart from the cellular to the whole organ level [ 8 , 9 ] . significant effort has been invested over the past decade in the field of mri cell tracking , in particular following the labeling of cells with either t1 or t2 shortening contrast agents . however , results of such studies have been mixed , indicating that cell labeling provides inconsistent assessment of cell survival . recent advances in mr reporter gene technology have enabled in vivo imaging of cell survival , migration , and differentiation primarily in stationary organs and tumors ( for a full review see ref ) . application of mr reporter gene imaging to the heart could transform cardiac regeneration research by enabling direct correlation of injected cell survival with important therapeutic outcomes such as restoration of contractile function and perfusion . paradoxically , studies of cell based cardiac regeneration therapy now number in the thousands , yet studies utilizing mr reporter gene imaging of cell survival following intramyocardial transplantation number in the single digits ( fig . research into the field of cardiac regeneration with cell therapy has undergone explosive growth since 1990 as evidenced by the large number of publications ( blue columns ) . an emerging consensus view considers in vivo imaging of cell fate decisions as a key tool for improving future therapeutic strategies . mri cell tracking following cell labeling with exogenous contrast agents ( red columns ) has emerged over the prior decade as a potential method by which to monitor the survival and viability of injected cells . more recently , the use of mri reporter genes ( green columns ) has been explored for in vivo imaging of cell survival , proliferation , and differentiation . in the context of mr reporter gene cell tracking in the heart , only four studies have to date been published on imaging the survival of reporter gene expressing cells following intramyocardial injection suever plot of relevant publications . research into the field of cardiac regeneration with cell therapy has undergone explosive growth since 1990 as evidenced by the large number of publications ( blue columns ) . an emerging consensus view considers in vivo imaging of cell fate decisions as a key tool for improving future therapeutic strategies . mri cell tracking following cell labeling with exogenous contrast agents ( red columns ) has emerged over the prior decade as a potential method by which to monitor the survival and viability of injected cells . more recently , the use of mri reporter genes ( green columns ) has been explored for in vivo imaging of cell survival , proliferation , and differentiation . in the context of mr reporter gene cell tracking in the heart , only four studies have to date been published on imaging the survival of reporter gene expressing cells following intramyocardial injection in this review we seek to discuss existing methods for mri cell tracking and the results of early studies of mr reporter gene cell tracking in the heart . given the small number of studies performed to date , each study is discussed in detail in order to highlight the important contributions made to the nascent field , and further questions that have arisen as a result of such studies . finally , we will discuss how important findings from mr reporter gene studies in stationary organs and tumors can potentially lead to more dynamic cmr reporter gene imaging . mri cell tracking has historically relied upon labeling of cells with chemical agents that generate contrast via modulation of either t1 ( bright contrast ) or t2 ( dark contrast ) relaxation times , or through selective excitation and transfer of saturated magnetization ( false color contrast ) . for example , labeling of fibroblasts with gadolinium - dtpa , a t1-shortening contrast agent , has been used to track the recruitment of fibroblasts to ovarian carcinoma tumors , and to track macrophage migration to the forming scar after myocardial infarction in the mouse heart . the recruitment of gadolinium labeled cells can either be visualized as positive contrast on heavily t1-weighted images , or can be measured by quantifying changes in tissue t1-relaxation times between areas with and without labeled cells . a more widely used cell labeling technique has involved the use of a large variety of iron oxide nanoparticles with a range of sizes and relaxivities . once internalized , iron oxide nanoparticles de - phase the surrounding magnetic field , leading to significantly shortened t2 relaxation times in the vicinity of labeled cells . labeled cells can then be easily tracked as signal voids on t2 * weighted images , or more quantitatively as areas of reduced t2-relaxation on t2-maps . while detection of cells labeled with t1 or t2 shortening contrast agents can be accomplished using conventional pulse sequences , the use of such agents , and in particular t2 shortening agents , can fundamentally limit the ability to image underlying anatomy or to combine cell tracking with additional measures of contractile function and perfusion . in addition concerns of asymmetric distribution of label in dividing cells , eventual dilution of the cell label , altered cell function following labeling , and in the case of iron oxide nanoparticles retention of label in damaged tissue long after the death of labeled cells [ 16 , 17 ] , all remain significant barriers to mri cell tracking with conventional contrast agents . an alternative cell labeling strategy that has recently emerged is to label cells with a class of contrast agents that selectively generate contrast through a process termed chemical exchange saturation transfer ( cest ) [ 1820 ] . following rf irradiation at a given cest agent s resonant frequency , the exchange of saturated magnetization with surrounding bulk water leads to a reduction of the on - resonance water signal . in juxtaposition to conventional t1/t2 shortening contrast agents , the contrast generated by cest agents can be selectively turned on . as a result , multiple cell populations that are labeled with distinct cest agents can be visualized in a multi - spectral manner , as was recently performed by ferrauto et al . . since the contrast from such labeling approaches does not fundamentally disturb the underlying anatomical integrity , cest - mri could be combined with additional measures of cardiac structure and function . mri reporter genes can uniquely overcome several of the barriers to conventional cell labeling strategies . first , when initiated with proper genetic reprogramming , mr reporter gene expression will remain constant across successive cell divisions . second , since transcription of the reporter gene occurs immediately after division , asymmetric division of an exogenous cell label is removed as a limiting factor to cell tracking . finally , since production of the mr reporter gene product ceases upon cell death , the likelihood of detecting a false positive signal after cell death is significantly reduced . while such characteristics are highly favorable , it is important to note that many of the first generation of mr reporter genes typically demonstrate lower sensitivity when compared to conventional cell labeling strategies . a large number of mr reporter gene strategies , each employing a different reporter mechanism , have been proposed and tested over the past decade ( for a full review see ref ) . overwhelmingly , three unique strategies have emerged and characterize the majority of mr reporter gene research ( fig . 2 ) . in the first strategy , over - expression of iron regulatory elements such as ferritin and/or the transferrin receptor act to increase cellular iron content , thereby shortening t2 relaxation times and enabling detection of reporter gene expressing cells on t2/t2 * weighted images . this strategy has been used to examine migration of reporter gene expressing cells in the brain [ 21 , 22 ] , the recruitment of reporter gene expressing cells to tumors , the proliferation of cancer cells , and differentiation of endothelial cells in the growing mouse embryo . the second broad strategy that has been pursued utilizes the expression of artificial plasma - membrane peptides or antigens as reporters ( for a full review see ref ) , with subsequent targeting using either t1 or t2 modulating contrast agents ( fig . 2 ) . finally , the third major strategy has been to mimic the prior strategies with mechanisms that selectively generate cest contrast through either constitutive expression of cest reporter genes , or through expression of transgenic kinases for targeted cest contrast agents . these strategies have largely been used to image cancer cell survival and proliferation following implantation in the rodent brain . of the more than 30 mr reporter gene studies published to date , only four have focused on dynamic cell tracking in the heart [ 28 , 29 , 30 , 31 ] , and are discussed in greater detail below.fig . mri reporter gene studies have overwhelmingly used three common strategies for in vivo detection of cell fate decisions . a large number of studies have examined over - expression or enhancement of iron regulatory elements including ferritin ( responsible for iron storage ) and/or the transferrin receptor ( responsible for shuttling of iron into cells ) . other studies have used expression of reporter genes encoding for artificial plasma membrane peptides or antigens with properly targeted iron oxide nanoparticles ( t2/t2 * contrast ) or gadolinium ( t1 contrast ) contrast agents . finally , a series of studies has examined expression of a class of engineered artificial reporter peptides that can selectively generate contrast on mr images following excitation with radiofrequency energy applied at reporter specific offset frequencies and chemical exchange of saturated magnetization ( cest ) with surrounding bulk water . in theory , such techniques could be combined in a single cell by placing each reporter gene downstream from a specific promoter ( p ) of interest . ( fe = iron , tfr = transferrin receptor , feo = iron oxide nanoparticles , gd = gadolinium chelates , = offset frequency , p = promoter of interest , m = plasma membrane targeted peptide , f = iron regulatory element of interest , c = artificial cest reporter gene ) common mri reporter gene mechanisms . mri reporter gene studies have overwhelmingly used three common strategies for in vivo detection of cell fate decisions . a large number of studies have examined over - expression or enhancement of iron regulatory elements including ferritin ( responsible for iron storage ) and/or the transferrin receptor ( responsible for shuttling of iron into cells ) . other studies have used expression of reporter genes encoding for artificial plasma membrane peptides or antigens with properly targeted iron oxide nanoparticles ( t2/t2 * contrast ) or gadolinium ( t1 contrast ) contrast agents . finally , a series of studies has examined expression of a class of engineered artificial reporter peptides that can selectively generate contrast on mr images following excitation with radiofrequency energy applied at reporter specific offset frequencies and chemical exchange of saturated magnetization ( cest ) with surrounding bulk water . in theory , such techniques could be combined in a single cell by placing each reporter gene downstream from a specific promoter ( p ) of interest . ( fe = iron , tfr = transferrin receptor , feo = iron oxide nanoparticles , gd = gadolinium chelates , = offset frequency , p = promoter of interest , m = plasma membrane targeted peptide , f = iron regulatory element of interest , c = artificial cest reporter gene ) mr reporter gene imaging is an exciting new field that is undergoing initial application to cardiac imaging . to date , cmr studies of reporter gene expressing cells have focused on monitoring cell viability and proliferation following engraftment into the rodent heart after surgically induced myocardial infarction . broadly , these studies have sought to identify reporter gene expressing cells through generation of t2/t2 * contrast on gradient echo images of the heart . in one series of studies , contrast was generated through over - expression of iron regulatory elements in order to safely increase intracellular iron content [ 28 , 30 , 31 ] . in a separate study , a reporter gene encoding for a plasma membrane bound antigen was targeted with intravenously delivered , antibody conjugated iron oxide nanoparticles [ 29 ] . these early studies have demonstrated a consistent ability to visually identify viable cell populations , confirmed continued expression of mr reporter genes following proliferation , and have not demonstrated any alterations in cell function as a result of reporter gene expression . in an early study by naumova et al . [ 30 ] murine skeletal myoblast cells ( c2c12 cell line ) were transduced to over - express mouse ferritin heavy chain in combination with the hemagglutinin acid ( ha ) tag under the control of the constitutively active cytomegalovirus ( cmv ) promoter . prussian blue staining of isolated cells confirmed enhanced uptake and retention of iron in c2c12 cells which over - expressed ferritin . the over - expression of ferritin did not affect cell viability or rates of cell division , however exposure of cells to ferric citrate ( fc ) significantly reduced the rate of cell division irrespective of ferritin over - expression . furthermore , over - expression did not change the differentiation patterns of c2c12 cells , as both normal and ferritin over - expressing c2c12 cells differentiated into multi - nucleated myotubes . initial in vitro phantom studies revealed a 25 % reduction of t2 relaxation times in ferritin over - expressing c2c12 cells when compared to normal c2c12 cells . in order to examine the impact of exposure to an iron rich environment , as would be the case in vivo , the authors incubated ferritin over - expressing cells in fc supplemented medium for 48 hours prior to phantom imaging . exposure to fc prior to imaging led to a further 50 % reduction of t2 relaxation times . in order to examine the in vivo utility of ferritin over - expression for mr cell tracking , the authors transplanted either c2c12 cells or ferritin over - expressing c2c12 cells into the hearts of c3h mice following surgically induced myocardial infarction ( fig . 3 ) . while initial differences in graft tissue signal intensity were minimal following transplantation , by 3 weeks after implantation ferritin over - expressing c2c12 grafts had accumulated sufficient iron to generate strong signal hypo - intensities on t2 * weighted images . in comparison , ex vivo staining protocols confirmed that both c2c12 and ferritin over - expressing c2c12 cells had differentiated into skeletal myoblasts replete with sarcomeric proteins . in a subsequent follow up study naumova and colleagues demonstrated that ferritin over - expressing cells were detected most easily when using standard bright - blood t2 * weighted gradient echo imaging , as opposed to black - blood gradient echo or proton density weighted spin echo imaging protocols . assessment of the size of ferritin over - expressing c2c12 grafts at 4 weeks after transplantation by mri correlated strongly with graft size as assessed by histology.fig . 3imaging cell survival and proliferation using ferritin over - expression . over - expression of ferritin heavy chain in mouse skeletal myoblasts ( c2c12 cells ) was used as an mr reporter gene strategy to safely increase cellular iron content and shorten t2/t2 * relaxation times . following intramyocardial injection , c2c12 cells that over - express ferritin heavy chain began to accumulate iron concomitant with the process of proliferation . within three weeks , sufficient iron had accumulated in ferritin over - expressing cells to enable detection as areas of hypo - intense signal using standard cardiac mr gradient echo imaging sequences ( red arrow , c2c12 + ferritin ) . cells that did not over - express ferritin heavy chain demonstrated similar signal intensity with surrounding myocardial tissue ( red arrow , c2c12 ) . histological assessment of isolated tissue sections confirmed increased iron deposition in grafts of c2c12 cells that over - expressed ferritin . [ 30 ] , but is reprinted with permission from imaging cell survival and proliferation using ferritin over - expression . over - expression of ferritin heavy chain in mouse skeletal myoblasts ( c2c12 cells ) was used as an mr reporter gene strategy to safely increase cellular iron content and shorten t2/t2 * relaxation times . following intramyocardial injection , c2c12 cells that over - express ferritin heavy chain began to accumulate iron concomitant with the process of proliferation . within three weeks , sufficient iron had accumulated in ferritin over - expressing cells to enable detection as areas of hypo - intense signal using standard cardiac mr gradient echo imaging sequences ( red arrow , c2c12 + ferritin ) . cells that did not over - express ferritin heavy chain demonstrated similar signal intensity with surrounding myocardial tissue ( red arrow , c2c12 ) . histological assessment of isolated tissue sections confirmed increased iron deposition in grafts of c2c12 cells that over - expressed ferritin . [ 30 ] , but is reprinted with permission from in a similar study , over - expression of human ferritin heavy chain was used as a reporter gene for mr tracking of swine cardiac stem cells following implantation into the infarcted rat heart . first used a lenti - virus to induce over - expression of human ferritin heavy chain , and subsequently cultured transgenic cardiac stem cells to form cardiospheres . in agreement with the findings of naumova et al . [ 30 , 31 ] , the authors found that over - expression of ferritin heavy chain did not affect cell viability or proliferation . further , the authors demonstrated that over - expression of human ferritin heavy chain did not change the expression levels of important cell surface markers of multi - potency and cardiac lineage . for in vivo rat studies , animals either underwent surgically induced myocardial infarction or sham surgery , and received injections of either phosphate buffered saline ( pbs ) , normal cardiospheres , or ferritin over - expressing cardio - spheres targeted to border zone tissue . the presence of ferritin heavy chain over - expressing cardiospheres was visualized using standard t2 * -weighted imaging as early as 1 week after implantation , and remained constant for 4 weeks after implantation in all rats that received ferritin over - expressing cardiospheres regardless of myocardial infarction status . in animals that underwent myocardial infarction surgery , injection of cardiospheres improved lv ejection fraction at four weeks after mi , with no differences observed between ferritin heavy chain over - expressing and wild type cardiospheres . prussian blue staining of isolated tissue sections confirmed elevated iron deposits in ferritin heavy chain over - expressing cardiospheres 4 weeks following implantation . importantly , repeated assessment after in vivo experiments of viability , proliferation , and key cell surface markers demonstrated no difference between ferritin heavy chain over - expressing and wild type cardiospheres . these promising early results demonstrate the ability to track the survival and proliferation of cells implanted into the healing infarct using ferritin over - expression as an mri reporter gene strategy . in both studies , ferritin over - expressing cells appeared as clear regions of signal voids on bright blood t2 * -weighted gradient echo images , mimicking the effect of cell labeling with iron oxide nanoparticles . interestingly , despite relatively similar quantities of injected cells , the signal voids generated by injection of c2c12 cells [ 30 , 31 ] were more pronounced than those generated following injection of cardiospheres . these small differences raise the important point that future studies should aspire to create a system by which ferritin over - expression generates consistent t2 * contrast across a wide array of candidate cells for cardiac regeneration . importantly , sufficient accumulation of iron in ferritin over - expressing grafts occurred in both studies , within 3 weeks when using c2c12 cells [ 30 , 31 ] and 1 week when using cardiospheres . the period required for sufficient iron accumulation falls well within the likely time between clinical visits for patients of future cell therapy trials . further , naumova and colleagues noted that unlike cell labeling strategies , over - expression of ferritin was maintained during the time - course of cell division and replication , leading to a reliable and consistent signal from surviving engrafted tissue [ 30 , 31 ] . it is important to note that imaging in both studies was performed on clinical scanners that required specialized small animal inserts . as a result , the spatial resolution of acquired images was lower that what is typically acquired in pre - clinical cell tracking studies , raising the possibility that estimation of graft size and cell proliferation from mr images could have been influenced by partial volume effects . acquisition of image data at higher spatial resolutions using dedicated small animal mr scanners , or performing similar studies in the hearts of larger animals could mitigate such concerns and further demonstrate the utility of ferritin over - expression as an mri reporter gene method for cardiac cell tracking . in contrast to the previously described studies which sought to generate t2/t2 * contrast via increased ferritin expression and subsequent accumulation of intercellular iron , an alternative mechanism has been to adapt immuno - histochemical staining techniques for use with mri . [ 29 ] engineered embryonic stem cells that express ha and myc antigens on the cell surface ( fig . 4 ) . while neither of these antigens independently function as mr specific reporters , chung et al . covalently bound super - paramagnetic iron oxide ( spio ) nanoparticles to monocloncal antibodies against ha and myc . initial in vitro studies demonstrated significant loss of signal in embryonic stem cells expressing ha and myc antigens when co - cultured with targeted spio particles that was not observed in non - transduced cells . later , in vivo studies demonstrated no difference in signal intensity of transplanted cells on t2 * -weighted images prior to administration of antibody bound spio particles . studies performed in animals receiving myc and ha positive embryonic stem cells and ha or myc targeted spio particles demonstrated reduced signal intensity in areas of injected cells starting at 5 days after cell injection , which was confirmed by whole body bioluminescence imaging . repeated studies revealed increased binding of targeted spio particles between five and ten days after cell implantation as the embryonic stem cells proliferated and began to form teratoma ( fig . similar studies performed with either falsely targeted or untargeted spio particles demonstrated minimal signal loss on t2 * -weighted images . myocardial tissue regions identified as containing large fractions of ha and myc expressing embryonic stem cells at mri were confirmed using h&e staining of isolated tissue sections . the use of targeted spio particles can overcome concerns of limited sensitivity that have been raised surrounding ferritin based reporter gene methods . in addition , the authors injected the targeted spio particles between 8 - 12 hours prior to imaging . this advantage suggests that upon translation , such agents could be injected outside of the scanner room and patients could return hours later for an mr examination . importantly , chung et al . [ 29 ] used a cell line known to induce teratoma formation , and did not characterize whether expression of the reporter gene construct altered cellular gene expression . when applied to additional pluripotent cell types , it will be critical to confirm that expressions of the reporter gene construct does not alter processes of cell viability , proliferation , and differentiation . further , as the authors acknowledge , expression of ectopic antigens may trigger an immune response that could destroy implanted cells when implanted into immuno - competent hosts.fig . 4imaging embryonic stem cell proliferation using over - expression of a plasma membrane antigen with targeted mri . ( a ) embryonic stem cells ( esc ) were engineered to express hemagglutinin acid ( ha ) and myc antigens at the plasma membrane . when super - paramagnetic iron oxide nanoparticles were properly targeted to ha and myc antigens , robust t2 * contrast could be generated in areas of reporter gene expressing cells . ( b ) ha and myc expressing esc were implanted into the infarct zone of the mouse heart following myocardial infarction and imaged at multiple time points . t2 * -weighted mr imaging of the heart after intravenous administration of the targeted iron oxide nanoparticles demonstrated signal voids in the presence of proliferating and teratoma forming esc ( white arrows ) . reporter gene expressing cells were also detected using bioluminescence imaging , which confirmed increased density of reporter gene expressing cells during teratoma formation . ( c d ) following administration of either false targeted spio - his - mab ( c ) or untargeted spio nanoparticles ( d ) , no significant loss of signal is seen in the myocardium of hearts receiving transplantation of ha and myc expressing esc . the presence of a clear signal on bioluminescence imaging confirms the presence of the esc and further confirms the selective targeting of ha and myc antigens . this material was reproduced with permission from john wiley & sons , inc . and chung et al . [ 29 ] imaging embryonic stem cell proliferation using over - expression of a plasma membrane antigen with targeted mri . ( a ) embryonic stem cells ( esc ) were engineered to express hemagglutinin acid ( ha ) and myc antigens at the plasma membrane . when super - paramagnetic iron oxide nanoparticles were properly targeted to ha and myc antigens , robust t2 * contrast could be generated in areas of reporter gene expressing cells . ( b ) ha and myc expressing esc were implanted into the infarct zone of the mouse heart following myocardial infarction and imaged at multiple time points . t2 * -weighted mr imaging of the heart after intravenous administration of the targeted iron oxide nanoparticles demonstrated signal voids in the presence of proliferating and teratoma forming esc ( white arrows ) . reporter gene expressing cells were also detected using bioluminescence imaging , which confirmed increased density of reporter gene expressing cells during teratoma formation . ( c d ) following administration of either false targeted spio - his - mab ( c ) or untargeted spio nanoparticles ( d ) , no significant loss of signal is seen in the myocardium of hearts receiving transplantation of ha and myc expressing esc . the presence of a clear signal on bioluminescence imaging confirms the presence of the esc and further confirms the selective targeting of ha and myc antigens . this material was reproduced with permission from john wiley & sons , inc . and chung et al . in an early study by naumova et al . [ 30 ] murine skeletal myoblast cells ( c2c12 cell line ) were transduced to over - express mouse ferritin heavy chain in combination with the hemagglutinin acid ( ha ) tag under the control of the constitutively active cytomegalovirus ( cmv ) promoter . prussian blue staining of isolated cells confirmed enhanced uptake and retention of iron in c2c12 cells which over - expressed ferritin . the over - expression of ferritin did not affect cell viability or rates of cell division , however exposure of cells to ferric citrate ( fc ) significantly reduced the rate of cell division irrespective of ferritin over - expression . furthermore , over - expression did not change the differentiation patterns of c2c12 cells , as both normal and ferritin over - expressing c2c12 cells differentiated into multi - nucleated myotubes . initial in vitro phantom studies revealed a 25 % reduction of t2 relaxation times in ferritin over - expressing c2c12 cells when compared to normal c2c12 cells . in order to examine the impact of exposure to an iron rich environment , as would be the case in vivo , the authors incubated ferritin over - expressing cells in fc supplemented medium for 48 hours prior to phantom imaging . exposure to fc prior to imaging led to a further 50 % reduction of t2 relaxation times . in order to examine the in vivo utility of ferritin over - expression for mr cell tracking , the authors transplanted either c2c12 cells or ferritin over - expressing c2c12 cells into the hearts of c3h mice following surgically induced myocardial infarction ( fig . 3 ) . while initial differences in graft tissue signal intensity were minimal following transplantation , by 3 weeks after implantation ferritin over - expressing c2c12 grafts had accumulated sufficient iron to generate strong signal hypo - intensities on t2 * weighted images . in comparison , grafts composed of normal c2c12 cells demonstrated normal signal intensity on identically weighted images . ex vivo staining protocols confirmed that both c2c12 and ferritin over - expressing c2c12 cells had differentiated into skeletal myoblasts replete with sarcomeric proteins . in a subsequent follow up study naumova and colleagues demonstrated that ferritin over - expressing cells were detected most easily when using standard bright - blood t2 * weighted gradient echo imaging , as opposed to black - blood gradient echo or proton density weighted spin echo imaging protocols . assessment of the size of ferritin over - expressing c2c12 grafts at 4 weeks after transplantation by mri correlated strongly with graft size as assessed by histology.fig . 3imaging cell survival and proliferation using ferritin over - expression . over - expression of ferritin heavy chain in mouse skeletal myoblasts ( c2c12 cells ) was used as an mr reporter gene strategy to safely increase cellular iron content and shorten t2/t2 * relaxation times . following intramyocardial injection , c2c12 cells that over - express ferritin heavy chain began to accumulate iron concomitant with the process of proliferation . within three weeks , sufficient iron had accumulated in ferritin over - expressing cells to enable detection as areas of hypo - intense signal using standard cardiac mr gradient echo imaging sequences ( red arrow , c2c12 + ferritin ) . cells that did not over - express ferritin heavy chain demonstrated similar signal intensity with surrounding myocardial tissue ( red arrow , c2c12 ) . histological assessment of isolated tissue sections confirmed increased iron deposition in grafts of c2c12 cells that over - expressed ferritin . [ 30 ] , but is reprinted with permission from imaging cell survival and proliferation using ferritin over - expression . over - expression of ferritin heavy chain in mouse skeletal myoblasts ( c2c12 cells ) was used as an mr reporter gene strategy to safely increase cellular iron content and shorten t2/t2 * relaxation times . following intramyocardial injection , c2c12 cells that over - express ferritin heavy chain began to accumulate iron concomitant with the process of proliferation . within three weeks , sufficient iron had accumulated in ferritin over - expressing cells to enable detection as areas of hypo - intense signal using standard cardiac mr gradient echo imaging sequences ( red arrow , c2c12 + ferritin ) . cells that did not over - express ferritin heavy chain demonstrated similar signal intensity with surrounding myocardial tissue ( red arrow , c2c12 ) . histological assessment of isolated tissue sections confirmed increased iron deposition in grafts of c2c12 cells that over - expressed ferritin . [ 30 ] , but is reprinted with permission from in a similar study , over - expression of human ferritin heavy chain was used as a reporter gene for mr tracking of swine cardiac stem cells following implantation into the infarcted rat heart . first used a lenti - virus to induce over - expression of human ferritin heavy chain , and subsequently cultured transgenic cardiac stem cells to form cardiospheres . in agreement with the findings of naumova [ 30 , 31 ] , the authors found that over - expression of ferritin heavy chain did not affect cell viability or proliferation . further , the authors demonstrated that over - expression of human ferritin heavy chain did not change the expression levels of important cell surface markers of multi - potency and cardiac lineage . for in vivo rat studies , animals either underwent surgically induced myocardial infarction or sham surgery , and received injections of either phosphate buffered saline ( pbs ) , normal cardiospheres , or ferritin over - expressing cardio - spheres targeted to border zone tissue . the presence of ferritin heavy chain over - expressing cardiospheres was visualized using standard t2 * -weighted imaging as early as 1 week after implantation , and remained constant for 4 weeks after implantation in all rats that received ferritin over - expressing cardiospheres regardless of myocardial infarction status . in animals that underwent myocardial infarction surgery , injection of cardiospheres improved lv ejection fraction at four weeks after mi , with no differences observed between ferritin heavy chain over - expressing and wild type cardiospheres . prussian blue staining of isolated tissue sections confirmed elevated iron deposits in ferritin heavy chain over - expressing cardiospheres 4 weeks following implantation . importantly , repeated assessment after in vivo experiments of viability , proliferation , and key cell surface markers demonstrated no difference between ferritin heavy chain over - expressing and wild type cardiospheres . these promising early results demonstrate the ability to track the survival and proliferation of cells implanted into the healing infarct using ferritin over - expression as an mri reporter gene strategy . in both studies , ferritin over - expressing cells appeared as clear regions of signal voids on bright blood t2 * -weighted gradient echo images , mimicking the effect of cell labeling with iron oxide nanoparticles . interestingly , despite relatively similar quantities of injected cells , the signal voids generated by injection of c2c12 cells [ 30 , 31 ] were more pronounced than those generated following injection of cardiospheres . these small differences raise the important point that future studies should aspire to create a system by which ferritin over - expression generates consistent t2 * contrast across a wide array of candidate cells for cardiac regeneration . importantly , sufficient accumulation of iron in ferritin over - expressing grafts occurred in both studies , within 3 weeks when using c2c12 cells [ 30 , 31 ] and 1 week when using cardiospheres . the period required for sufficient iron accumulation falls well within the likely time between clinical visits for patients of future cell therapy trials . further , naumova and colleagues noted that unlike cell labeling strategies , over - expression of ferritin was maintained during the time - course of cell division and replication , leading to a reliable and consistent signal from surviving engrafted tissue [ 30 , 31 ] . it is important to note that imaging in both studies was performed on clinical scanners that required specialized small animal inserts . as a result , the spatial resolution of acquired images was lower that what is typically acquired in pre - clinical cell tracking studies , raising the possibility that estimation of graft size and cell proliferation from mr images could have been influenced by partial volume effects . acquisition of image data at higher spatial resolutions using dedicated small animal mr scanners , or performing similar studies in the hearts of larger animals could mitigate such concerns and further demonstrate the utility of ferritin over - expression as an mri reporter gene method for cardiac cell tracking . in contrast to the previously described studies which sought to generate t2/t2 * contrast via increased ferritin expression and subsequent accumulation of intercellular iron , an alternative mechanism has been to adapt immuno - histochemical staining techniques for use with mri . [ 29 ] engineered embryonic stem cells that express ha and myc antigens on the cell surface ( fig . 4 ) . while neither of these antigens independently function as mr specific reporters , chung et al . covalently bound super - paramagnetic iron oxide ( spio ) nanoparticles to monocloncal antibodies against ha and myc . initial in vitro studies demonstrated significant loss of signal in embryonic stem cells expressing ha and myc antigens when co - cultured with targeted spio particles that was not observed in non - transduced cells . later , in vivo studies demonstrated no difference in signal intensity of transplanted cells on t2 * -weighted images prior to administration of antibody bound spio particles . studies performed in animals receiving myc and ha positive embryonic stem cells and ha or myc targeted spio particles demonstrated reduced signal intensity in areas of injected cells starting at 5 days after cell injection , which was confirmed by whole body bioluminescence imaging . repeated studies revealed increased binding of targeted spio particles between five and ten days after cell implantation as the embryonic stem cells proliferated and began to form teratoma ( fig . similar studies performed with either falsely targeted or untargeted spio particles demonstrated minimal signal loss on t2 * -weighted images . myocardial tissue regions identified as containing large fractions of ha and myc expressing embryonic stem cells at mri were confirmed using h&e staining of isolated tissue sections . the use of targeted spio particles can overcome concerns of limited sensitivity that have been raised surrounding ferritin based reporter gene methods . in addition , the authors injected the targeted spio particles between 8 - 12 hours prior to imaging . this advantage suggests that upon translation , such agents could be injected outside of the scanner room and patients could return hours later for an mr examination . importantly , chung et al . [ 29 ] used a cell line known to induce teratoma formation , and did not characterize whether expression of the reporter gene construct altered cellular gene expression . when applied to additional pluripotent cell types , it will be critical to confirm that expressions of the reporter gene construct does not alter processes of cell viability , proliferation , and differentiation . further , as the authors acknowledge , expression of ectopic antigens may trigger an immune response that could destroy implanted cells when implanted into immuno - competent hosts.fig . 4imaging embryonic stem cell proliferation using over - expression of a plasma membrane antigen with targeted mri . ( a ) embryonic stem cells ( esc ) were engineered to express hemagglutinin acid ( ha ) and myc antigens at the plasma membrane . when super - paramagnetic iron oxide nanoparticles were properly targeted to ha and myc antigens , robust t2 * contrast could be generated in areas of reporter gene expressing cells . ( b ) ha and myc expressing esc were implanted into the infarct zone of the mouse heart following myocardial infarction and imaged at multiple time points . t2 * -weighted mr imaging of the heart after intravenous administration of the targeted iron oxide nanoparticles demonstrated signal voids in the presence of proliferating and teratoma forming esc ( white arrows ) . reporter gene expressing cells were also detected using bioluminescence imaging , which confirmed increased density of reporter gene expressing cells during teratoma formation . ( c d ) following administration of either false targeted spio - his - mab ( c ) or untargeted spio nanoparticles ( d ) , no significant loss of signal is seen in the myocardium of hearts receiving transplantation of ha and myc expressing esc . the presence of a clear signal on bioluminescence imaging confirms the presence of the esc and further confirms the selective targeting of ha and myc antigens . this material was reproduced with permission from john wiley & sons , inc . and chung et al . [ 29 ] imaging embryonic stem cell proliferation using over - expression of a plasma membrane antigen with targeted mri . ( a ) embryonic stem cells ( esc ) were engineered to express hemagglutinin acid ( ha ) and myc antigens at the plasma membrane . when super - paramagnetic iron oxide nanoparticles were properly targeted to ha and myc antigens , robust t2 * contrast could be generated in areas of reporter gene expressing cells . ( b ) ha and myc expressing esc were implanted into the infarct zone of the mouse heart following myocardial infarction and imaged at multiple time points . t2 * -weighted mr imaging of the heart after intravenous administration of the targeted iron oxide nanoparticles demonstrated signal voids in the presence of proliferating and teratoma forming esc ( white arrows ) . reporter gene expressing cells were also detected using bioluminescence imaging , which confirmed increased density of reporter gene expressing cells during teratoma formation . ( c d ) following administration of either false targeted spio - his - mab ( c ) or untargeted spio nanoparticles ( d ) , no significant loss of signal is seen in the myocardium of hearts receiving transplantation of ha and myc expressing esc . the presence of a clear signal on bioluminescence imaging confirms the presence of the esc and further confirms the selective targeting of ha and myc antigens . this material was reproduced with permission from john wiley & sons , inc . and chung et al . as the field of cardiac mri reporter gene based cell tracking continues to develop , there are several key objectives that must be part of the field s evolution . first , the need to genetically alter cells in order to express mr reporter genes remains a barrier to translation beyond pre - clinical models . although early mr reporter gene studies have demonstrated minimal impact on cell viability , proliferation , function , and differentiation , and newer site specific integration techniques for genetic modification of cells demonstrate superb safety profiles , thorough examination of the effects of mr reporter gene expression on cell function and proliferation must be performed in long term studies to exclude potentially negative outcomes . second , mr reporter gene cell tracking in the heart has only used constitutive expression of reporter genes . as the field of cell therapy seeks to promote differentiation of pluripotent cells into a variety of cells including cardiomyocytes and endothelial cells , it will be important to test the ability of mr reporter genes to enable visualization of cell differentiation along different lineages . finally , amongst the studies performed to date , all have used mr reporter genes that generate loss of signal on t2 * weighted images . ideally , various mr reporter gene strategies ( cest , iron regulatory elements , and/or plasma membrane antigens with targeted t1 and t2 * contrast agents ) could be combined to elucidate a more complete picture of molecular processes within cells , or to examine the differentiation of stem cells into a variety of terminal cell types by linking each reporter gene to cell type specific promoters . mr reporter gene imaging is an exciting and burgeoning field that can potentially enable a multi - scale understanding of how cell fate decisions impact subsequent disease and regenerative processes . however , as this field continues to evolve , the potential to elucidate multiple aspects of cell fate decisions within the framework of cardiac regeneration becomes increasingly possible . with further development , mr reporter gene imaging can someday be used to investigate multiple aspects of cell survival , proliferation , and differentiation in order to gain a deeper understanding of mechanisms by which to promote stem cell survival and regeneration of myocardial tissue .

we recently reported the safety , immunological and clinical responses to a gpc3-derived peptide vaccine in a phase i clinical trial of patients with advanced hepatocellular carcinoma ( hcc ) . we conducted a subsequent trial in advanced hcc to assess the histopathological findings before and after vaccination with the gpc3 peptide . here , we present the clinical course and the pathological study including the autopsy of a patient with advanced hcc in the ongoing clinical trial . a 62-year old patient suffering from hcc refractory to sorafenib therapy received the gpc3 peptide vaccine . the patient had fever and remarkably impaired liver function twice after vaccination . contrast - enhanced ct after the second vaccination showed multiple low - density areas in the liver tumor , indicating tumor necrosis . in contrast , the tumor thrombus in the right atrium increased . the patient discontinued protocol treatment due to disease progression and died 30 days after the second vaccination . an autopsy was performed to determine the main cause of death and to evaluate the antitumor effect of the vaccination . a histological examination showed central necrosis in most of the intrahepatic tumor . the main cause of death was circulatory failure due to tumor thrombus , which occupied most of the right atrium . an immunohistochemical analysis revealed infiltration of cd8-positive t cells in the residual carcinoma , but not within the cirrhotic area . ex vivo ifn- enzyme - linked immunospot analysis revealed vaccine - induced immune - reactivity against the gpc3 peptide . a histopathological examination at the estimated time of a strong immunological response demonstrated a gpc3 peptide vaccination - induced cytotoxic t - lymphocyte response with an anti - tumor effect . research into cell therapy based cardiac repair and regeneration has experienced explosive growth over the last decade , however further progress is hindered by an inability to serially and non - invasively image cell survival and fate decisions following implantation . recent advances in magnetic resonance imaging ( mri ) reporter gene techniques have enabled in vivo imaging of cell survival , proliferation , migration , and differentiation , however this has mostly been performed in stationary tissues . a small series of recent studies has examined the possibility of using mri reporter genes to track the survival of cells injected into the heart following myocardial infarction . in this review , we seek to frame the emerging field of mri reporter gene based cardiac cell tracking within the larger framework of the needs of cardiac regeneration therapy and the more established field of mri cell tracking . while initial studies have demonstrated a promising ability to track the viability and proliferation of cells used for cell therapy , the ultimate goal of mr reporter gene imaging in the heart remains the ability to simultaneously correlate cell fate decisions with additional measures of structural and functional recovery .

olefin metathesis is a highly versatile tool for the generation of carbon carbon bonds , and a variety of applications have evolved around its implementation . the broad utility of olefin metathesis is a consequence of the exceptional selectivity , activity , and functional - group compatibility of select metathesis catalysts , highlighted by carbon carbon bond formation on a variety of complex substrates including small molecules , natural products , organic and inorganic materials , and even proteins . the use of metathesis in biological applications is an emerging field of research , in part , because of advances in the genetic and chemical incorporation of alkene - containing amino acids into peptides and proteins . carbon bonds with high fidelity for applications in peptide stapling , as surrogates of hydrogen bonding , and as methods for modifying peptides and proteins used to mimic physiologically relevant post - translational modifications . the application of metathesis for stabilizing peptide secondary structure and for performing selective protein modifications has implications for imparting greater metabolic stability , cellular permeability , and higher binding affinity toward biological targets . peptides used as inhibitors of hiv fusion and assembly , as modulators of signaling pathways involved in cancer , and as selective activators of enzymes involved in diabetes . despite the tremendous success of metathesis in peptide and peptidomimetic research , the ability to control olefin geometry in the product has been met with limited success . most metathesis catalysts exhibit minimal kinetic selectivity , and thus , the product distribution reflects the thermodynamic stability of each olefin isomer . in many cases , a mixture of e and z isomers this imposes challenges for examining the influence of alkene geometry on the stability and activity of diverse compounds . in pursuit of catalysts with greater control over olefin products , we uncovered a series of cyclometalated ruthenium catalysts that could achieve high conversions with exquisite z selectivity ( figure 1 ) . the origin of z selectivity for cyclometalated ruthenium catalysts involves approach of the olefin from a side - bound position ( i.e. , cis to the nhc ligand and trans to the chelating adamantyl ) and is favored through a combination of steric and electronic effects imposed by the nhc ligand . although catalysts 1 and 2 demonstrate excellent selectivity in olefin metathesis , their activity on complex substrates , including peptides , remained unexplored . to this end , we sought to initiate a comprehensive evaluation of z - selective metathesis on peptides using newly developed cyclometalated ruthenium catalysts . through the combined efforts of homodimerization , cross metathesis and ring - closing metathesis , we have developed guidelines for assessing the influence of amino acids and peptides on catalyst activity and selectivity . these principles were applied for carrying out z - selective metathesis on challenging substrates including peptides that comprise parallel -sheets and on stapling of -helical peptides . our goal for expanding the utility of z - selective ruthenium catalysts was to examine the influence of amino acids on the selectivity and activity of catalysts 1 and 2 . catalysts bearing n - adamantyl substituents and bidentate nitrato ligands were found to be critical for achieving high z selectivity , and we set out to determine whether such catalysts could be applied to substrates bearing multiple functionalities and with varying steric profiles . to benchmark the reactivities of catalysts 1 and 2 , we chose to investigate the homodimerization of protected amino acids modified with homoallyl functionality ( table 1 ) . our initial studies focused on the use of alanine 3 , as we anticipated that amino acids bearing unhindered and aliphatic side chains would provide an ideal platform for comparative studies . we began with a catalyst loading of 2.5 mol % in tetrahydrofuran ( thf ) at 40 c , and this afforded the homodimerization product 4 in 53% yield after 4 h using catalyst 1 and 58% yield in the presence of catalyst 2 ( entry 1 ) . gratifyingly , the z selectivity remained high ( 90% ) throughout the course of the reaction . catalyst loadings of 5 mol % afforded product 4 in 65% yield with 92% z selectivity in the presence of catalyst 2 ( entry 2 ) . increasing the concentration led to modest improvements in yields while maintaining high z selectivity ( entries 3 and 4 ) . ultimately , we found that using a catalyst loading of 7.5 mol % afforded product 4 in 76% yield with 94% z selectivity ( entry 6 ) , and these conditions proved to be optimal among the various reaction conditions explored . we next examined the solvent dependence of the activities of catalysts 1 and 2 in homodimerization , as this is an important consideration in view of the solubility profiles of many peptidic substrates ( table 2 ) . several polar and nonpolar solvents were investigated reflecting those most often used in peptide synthesis . coordinating solvents [ e.g. , acetonitrile ( mecn ) ] were less active in promoting z - selective metathesis as compared to noncoordinating solvents [ e.g. , dicholorethane ( dce ) ] ( entries 2 and 3 ) . polar solvents including dimethylformamide ( dmf ) , dimethyl sulfoxide ( dmso ) , and n - methylpyrrolidone ( nmp ) were tolerated by catalysts 1 and 2 , affording product 4 in yields ranging from 55% to 67% and 90% z selectivity ( entries 46 ) . protic solvents including methanol ( meoh ) , ethanol ( etoh ) , and aqueous tert - butanol ( t - buoh ) mixtures yielded highly enriched z - olefin products ( entries 79 ) . other polar solvents including nitromethane ( meno2 ) resulted in low conversions ( entry 10 ) , presumably by activating decomposition pathways of the cyclometalated ruthenium catalysts . it is worth noting that , across the variety of reaction conditions explored , the z selectivity remained consistently high . determined by h or c nmr spectroscopy . determined by h or c nmr spectroscopy . to probe the activities of catalysts 1 and 2 further , we investigated the homodimerization of other canonical amino acids ( table 3 ) . boc - protected amino acids bearing aliphatic or aromatic side chains were active in z - selective metathesis ( entries 14 ) . branched side chains from amino acids including valine ( 5a ) , isoleucine ( 5b ) , and leucine ( 5c ) afforded products 6a c in yields approaching 74% with 94% z selectivity ( entries 13 ) . aromatic side chains from amino acids including phenylalanine 5d afforded product 6d in 75% yield and 93% z selectivity in the presence of catalyst 2 ( entry 4 ) . exceptions include glycine and proline , as these are inactive in homodimerization ( entries 5 and 6 ) . homodimerization of tryptophan afforded product 6 g in 64% yield with 90% z selectivity ( entry 7 ) . in contrast , histidine was deemed incompatible with catalysts 1 and 2 ( entry 8) . unprotected alcohols from the side chains of serine 5i or threonine 5j were tolerated by catalysts 1 and 2 but required protection to reach acceptable conversions , providing products 6i and 6j in 72% and 73% yields , respectively ( entries 9 and 10 ) . homodimerization of tyrosine afforded product 6k in 64% yield and 87% z selectivity in the presence of catalyst 1 and 68% yield and 90% z selectivity using catalyst 2 ( entry 11 ) . in contrast to alcohols , side chains bearing thiols ( i.e. , cysteine ) or thioethers ( i.e. , methionine 5l ) generally led to catalyst deactivation ( entry 12 ) . protecting the side chain of cysteine could overcome catalyst inactivity and afford product 6 m in 55% yield and 87% z selectivity ( entry 13 ) . polar side chains bearing carboxylate ( 5n , o ) , carboxamide ( 5p , q ) , or amine ( 5r ) functionality required protection prior to homodimerization . in these cases , yields from 70% to 80% could be achieved with high z selectivity ( entries 1418 ) . side chains bearing a protected guanidinium functionality ( i.e. , arginine 5s ) were less active in homodimerization , affording product 6s in 34% and 33% yields using catalysts 1 and 2 , respectively . these findings reveal that the identity of the amino acid side chain can profoundly influence the activity of cyclometalated ruthenium catalysts . determined by h or c nmr spectroscopy . side chain protected with a trityl group . emboldened by the success of z - selective homodimerization of amino acids , we next evaluated the effects of varying the metathesis partner to achieve z - selective cross metathesis ( cm ) ( table 4 ) . for our studies , we chose cross partners that showed variable activity in homodimerization . in this way , we could begin to classify substrates on the basis of their propensity for achieving productive cm . we explored cm using homoallyl - modified alanine 3 and the similarly reactive substrate valine 5a as the cross partner . cross metathesis in the presence of an equimolar ratio of 3 to 5a afforded the desired heterocross product 7 in 41% yield with 90% z selectivity ( entry 1 ) . increasing the stoichiometry of 5a relative to 3 led to a modest increase in yield , providing product 7 in 48% yield with 91% z selectivity ( entry 2 ) . further increases in 5a afforded similar trends , with yields approaching 60% and 90% z selectivity ( entries 3 and 4 ) . to demonstrate the versatility of this method , we explored conditions in which 5a was used as the limiting reagent ( entries 57 ) . under these conditions , an incremental increase in the yield of product 7 was observed upon increasing the ratio of 3 to 5a . as in the case of excess 5a , modest improvements in the yield of 7 were achieved with increasing equivalents of 3 , demonstrating the statistical nature of cm using similar reactive substrates . given the modest conversions to product and high z selectivities , we can conclude that the products of cm are sparingly consumable using catalysts 1 and 2 , ensuring that the z selectivity can remain high throughout the course of the reaction . we next evaluated whether amino acids of differing reactivity profiles could be used for selective cm ( table 5 ) . in choosing the requisite cross partners , we focused on substrate 3 and homoallyl - modified arginine 5s , as they both showed relatively low reactivity in homodimerization . we began our studies using an equimolar ratio of 3 and 5s , and this afforded 8 in 46% yield and 93% z selectivity ( entry 1 ) . increasing the ratio of 5s to 3 led to slightly diminished yields and z selectivities ( entries 24 ) . by contrast , reversing the order such that 3 was in excess of 5s afforded 8 in yields of 47% and 58% using catalysts 1 and 2 , respectively ( entry 5 ) . further increasing the ratio of 3 to 5s could achieve the heterocross product 8 in improved yields and high z selectivities ( entries 6 and 7 ) . taken together , these findings reveal that the intrinsic reactivity differences between homoallyl - modified amino acids can be used for productive z - selective cross metathesis . the unique reactivity profiles of canonical amino acids in z - selective homodimerization and cm prompted investigation of a subset of non - natural amino acids that have shown promise in peptide and protein modification using olefin metathesis . allyl - protected amino acids including serine , cysteine , and selenocysteine have been shown to enhance the rate of metathesis when incorporated into peptides and proteins . studies by davis and co - workers ascribed the unique chemical reactivity of such amino acids through a chelation - assisted mechanism whereby precoordination of the heteroatom to ruthenium increases the effective concentrations of the alkylidene and alkene without detrimental chelation . moreover , softer nucleophiles such as sulfur and selenium were found to have an activating effect relative to oxygen for enhancing the rate of cm . these findings are intriguing considering that sulfur can have a deactivating effect on olefin metathesis , and our results suggest that sulfur - containing amino acids ( e.g. , compound 5l ) lead to catalyst inactivity . nonetheless , a wealth of information suggests that heteroatoms can modulate metathesis activity ; however , this phenomenon was unexplored using z - selective cyclometalated catalysts . to investigate the influence of allylic heteroatoms on the activities of catalysts 1 and 2 , we examined a series of allyl - protected amino acids in homodimerization ( table 6 ) . we chose allylglycine ( 9a ) and homoallylglycine ( 9b ) , as well as allyl - protected serine ( 9c ) and cysteine ( 9d ) , for our studies . in this sense , 9a d would reveal both the role of sterics ( i.e. , comparison of 9a and 9b ) and the effect of chelation by heteroatoms ( i.e. , comparison of 9b to 9c , d ) in facilitating metathesis . for these tests , we investigated homodimerization of 9a and compared its reactivity relative to that of substrate 9b . conversion of 9a to the dimerized product 10a was low using catalyst 1 or 2 , occurring in 45% yield and 90% z selectivity ( entry 1 ) . by comparison , dimerization of homoallylglycine 9b afforded product 10b in 59% yield and 90% z selectivity ( entry 2 ) . this corroborates our earlier findings that the steric environment around the alkene can influence the efficiency of z - selective metathesis . allyl - protected serine 9c afforded product 10c in 67% yield with 92% z selectivity ( entry 3 ) . by comparison , cysteine 9d was more active in homodimerization , leading to 71% yield and 93% z selectivity in the presence of catalyst 2 ( entry 4 ) . these results suggest that allylic heteroatoms can facilitate z - selective metathesis in the presence of cyclometalated ruthenium catalysts . determined by h or c nmr spectroscopy . the insights garnered from the homodimerization of substrates 9a d revealed that the identity of the alkene can influence the activities of catalysts 1 and 2 in z - selective metathesis . although these studies provide important insight for assessing the intrinsic reactivity of allyl - modified amino acids with cyclometalated ruthenium catalysts , the general utility of such catalysts could be further illuminated by their use in cm . as such , we examined catalysts 1 and 2 in cm using allyl - modified amino acids 9a d ( table 7 ) . to assess the relative activities of substrates 9a d in cm , we chose allyl acetate 11 as the cross partner , as we previously showed that 11 is highly active in z - selective cm . we explored a variety of conditions to test the generality of cm including the use of solvents that are compatible with native peptides and proteins . for our initial studies , we examined cm between allylglycine 9a and 11 under our previously optimized conditions , and this afforded the heterocross product 12a in 42% yield and 90% z selectivity . we next explored cm under aqueous conditions , including the use of additives shown to enhance the efficiency of cm on peptides and proteins . in the presence of aqueous tert - butanol , the heterocross product 12a was achieved in 38% yield with 90% z selectivity ( entry 2 ) . inclusion of salts such as licl or mgcl2 , which has been shown to be beneficial for enhancing methathesis on peptides , afforded product 12a in 31% yield but with diminished z selectivity ( entries 3 and 4 ) . these trends were also observed using homoallylglycine 9b as the cross partner ( entries 58 ) . to investigate whether amino acids bearing allylic heteroatoms influence the efficiency of z - selective cm , we exposed allylserine 9c and allylcysteine 9d to similar reaction conditions . synthesis of the heterocross product was improved , affording 12c in 63% yield with 92% z selectivity using catalyst 2 ( entry 9 ) . the use of aqueous conditions ( entry 10 ) or inclusion of additives ( entries 11 and 12 ) led to slightly diminished yields and z selectivity . by comparison , the use of allylcysteine 9d as the cross partner afforded the desired product 12d in 60% yield with 90% z selectivity ( entry 13 ) . as observed with substrates 9a c , a decrease in catalyst activity occurred under aqueous conditions and in the presence of salt additives ( entries 1416 ) . collectively , these results suggest that the activities of catalysts 1 and 2 are highly dependent on the reaction conditions . in general , the trends observed in cm with 9a d parallel those observed using nonchelated ruthenium catalysts ; however , the use of salts as additives appears to have a deactivating effect on the activity of cyclometalated ruthenium catalysts . these results lend support to the importance of the chelating ligand ( i.e. , bidentate versus mondentate ligand coordination ) in the activities and selectivities of catalysts 1 and 2 . determined by h or c nmr spectroscopy . reaction conditions : 1 mmol of 9a d in 1:1 h2o / t - buoh . reaction conditions : 1 mmol of 9a d in 1:1 h2o / t - buoh + 2 mm licl . reaction conditions : 1 mmol of 9a d in 1:1 h2o / t - buoh + 2 mm mgcl2 . investigation of both canonical and noncanonical amino acids in homodimerization and cross metathesis revealed that the choice of cross partner is critical to the success of ruthenium - catalyzed z - selective metathesis . in this regard , amino acid side chains bearing aliphatic , aromatic , or protected polar functionalities were highly active in metathesis . those amino acids that generally lead to lower conversion ( i.e. , arginine ) could undergo selective cross metathesis in the presence of a more highly reactive substrate . moreover , the steric environments around the olefin and allylic heteroatoms were shown to impact the efficiency of z - selective cm . whereas these observations apply generally to amino acids that are commonly used in olefin metathesis , we wanted to investigate whether cyclometalated ruthenium catalysts could be used in cm on more complex substrates , including peptides . in choosing the requisite cross partners , we took inspiration from peptides known to adopt defined -sheet secondary structures when tethered through a turn - promoting moiety or as part of a macrocycle . such structures hold promise in applications ranging from supramolecular chemistry to biology and offer challenging substrates for catalysts 1 and 2 . a wealth of information regarding the use of peptides and small molecules as -sheet mimics has revealed that both hydrogen bonding and amino acid side - chain pairing preferences can be used to dictate the stability of -sheet formation . to this end , we synthesized peptides 13 and 14 , which represent typical sequences found in parallel -sheets and whose structures are known to rely on the sequence of amino acids ( scheme 1 ) . for our studies , we used an excess of peptide 13 , as we had shown that less reactive amino acids , including arginine , could be used for selective cross metathesis in the presence of more highly active amino acids . under these conditions , conversion reached 60% for the desired peptide 15 with greater than 90% z selectivity . attempts to improve the yield of the heterocross product by conducting cm in solvents that promote hydrogen bonding and thereby preorganize the peptides to facilitate metathesis led to similar conversions . these results attest to the functional - group tolerability of cyclometalated ruthenium catalysts and point to further strategies aimed at accessing complex olefinic substrates bearing multiple functionalities . determined by analytical high - performance liquid chromatography mass spectrometry ( hplc - ms ) . our results regarding the activity of cyclometalated ruthenium catalysts in cross metathesis of amino acids and peptides revealed that the kinetic selectivities of catalysts 1 and 2 can be used to synthesize peptides highly enriched in z - olefins . although these studies provide a framework for promoting metathesis through judicious choice of the amino acid sequence , we observed that conversions in cm were optimal at relatively high concentrations ( 0.30.4 m ) , which , for many substrates , might be a limitation . we sought a more general strategy such that a variety of reaction conditions could be used to promote z - selective metathesis on a diverse collection of peptides . to this end , we investigated ring - closing metathesis ( rcm ) of olefinic amino acids for the synthesis of stapled peptides . such peptides hold promise as novel therapeutics by virtue of their enhanced -helicity ; proteolytic stability ; and ability to target intracellular proteins involved in cancer , infectious disease , and metabolism . as such , we set about to broaden the available catalysts used to synthesize this important class of peptides under conditions that would be amenable to comprehensive screening of catalyst activity in the presence of varying peptide sequences . traditional methods for the synthesis of stapled peptides by rcm have relied on the use of o - allyl serine or c-tetrasubstituted amino acids to install macrocyclic cross - links into synthetic peptides . most strategies incorporate non - natural amino acids at positions spanning one ( i , i + 4 ) or two ( i , i + 7 ) turns of a helix that serve to preorganize the reactive side chains on the same helical face . a wealth of knowledge derived from computational and experimental approaches has illuminated the minimal constraints necessary for achieving rcm on peptides using first- and second - generation or grubbs an unmet challenge in the synthesis of stapled peptides has been the ability to control olefin geometry in the product , as the use of noncyclometalated ruthenium catalysts typically gives rise to both e and z isomers that are often inseparable . this imposes challenges for examining the role of olefin geometry in the stability and biological activity of stapled peptides , which , to date , has not been thoroughly explored . to this end , we employed catalysts 1 and 2 in z - selective rcm for stapling -helical peptides that encompass the vast majority of peptides used for biological studies . as part of our ongoing effort to expand the utility of catalysts 1 and 2 , we chose to conduct rcm on resin - supported peptides . this would streamline the synthesis of peptides and offer a modular platform to test the activities of cyclometalated ruthenium catalysts . our goal was to compare the activities of z - selective catalysts with those of noncyclometalated ruthenium catalysts in rcm , and we focused our efforts on peptides with known biological activity . the sequence we chose is derived from an -helical peptide known to target the bcl-2 family of proteins involved in the regulation of apoptosis and whose activity is modulated by constraining the peptide through hydrocarbon stapling ( table 8) . the chemical features of peptide 16 consist of two stereochemically defined ,-disubstituted olefinic amino acids separated by one turn of a helix ( i.e. , olefins positioned at residues i and i + 4 ) that , upon ring closure , would generate a 21-membered macrocycle . loading capacities for resin : wang , 0.5 mmol / g ; tentagel , 0.25 mmol / g ; mbha , 0.5 mmol / g . amino acids were protected prior to rcm . determined by analytical hplc - ms . the stabilities and activities of catalysts 1 and 2 in the presence of resins were unexplored , and we examined commonly used resins for solid - phase peptide synthesis that varied based on composition and loading capacity . throughout our studies , we performed rcm in solvents that promote -helicity [ e.g. , dicholorethane ( dce ) ] at concentrations that favor macrocyclic ring closure . initial screening revealed that the choice of resin influenced the activities of catalysts 1 and 2 in rcm . conversions to the desired rcm product 17 were typically low on wang resin using 10 mol % catalyst at room temperature for 2 h ( entry 1 ) . resins bearing hydrophilic linkers proved beneficial , affording conversions approaching 40% under the same reaction conditions ( entry 2 ) . the use of mbha resin led to 60% conversion ( entry 3 ) , and we focused further optimization using this resin . we next explored the effects of reaction time and catalyst loading on rcm . prolonging the reaction led to modest improvements , generating product 17 in 70% conversion with greater than 90% z selectivity ( entry 4 ) , and subjecting the resin - bound peptide to successive rounds of catalyst resulted in conversions of 75% using two cycles of catalyst addition ( entry 5 ) . increasing the temperature to 40 c afforded 17 in 80% yield and with greater than 90% z selectivity ( entry 6 ) . to probe the generality of the method , we investigated peptides bearing olefinic amino acids spanning two turns ( i , i + 7 ) of a helix with varying amino acid sequences ( scheme 2 ) . to span the distance of two helical turns , we modified the n - terminal olefinic amino acid by increasing the tether length ( from five to eight carbon atoms ) and inverting the stereochemical configuration ( from s to r ) , both of which were predicted to facilitate rcm . under our optimized conditions , conversions of 85% for the desired rcm product 19 could be achieved after two cycles of catalyst addition with greater than 90% z selectivity . these results demonstrate that cyclometalated ruthenium catalysts can promote z - selective rcm on solid support for the synthesis of stapled peptides bearing all hydrocarbon cross - links . determined by analytical hplc - ms . we earlier demonstrated that aib - rich peptides bearing i , i + 3 l - serine o - allyl residues afford highly e - selective rcm products , in studies motivated , in part , by a theoretical prediction that suggested an rcm - derived 18-membered ring using these side chains would serve as a minimal constraint for the 310-helix . we were curious to see whether a z - selective catalyst could overcome any substrate bias favoring the e - olefin geometry . to this end , we studied the rcm conversions of pentapeptide boc - l - ser(al)-aib - aib - l - ser(al)-aib - ome ( 20 ) and heptapeptide boc - l - val - l - ser(al)-l - leu - aib - l - ser(al)-l - val - l - leu - ome ( 24 ) to macrocycles 21 and 25 , respectively , using second - generation catalysts 22 and 23 and z - selective catalyst 1 ( table 9 ) . as expected from our earlier studies , peptide 20 readily cyclized to the e macrocycle in the presence of 10 mol % 22 or 23 in dce held at 45 c for 10 h ( entries 1 and 2 ) . under similar reaction conditions , no macrocyclization was observed with catalyst 1 , even with a 3-fold increase in catalyst loading and extended reaction time ( entry 3 ) . the same behavior was observed for heptapeptide 24 , although , in this case , the z macrocycle did form with 22 or 23 to the extent of ca . 8% ( entries 4 and 5 ) . peptide 24 rapidly cyclized with the second - generation catalysts in refluxing dichloromethane , but it was unreactive toward catalyst 1 under identical conditions ( entry 6 ) . whereas most peptide side - chain rcm reactions produce e / z mixtures , the minimal i , i + 3 cross - link in these aib - containing systems seems reluctant to form the z - olefin , probably a consequence of the conformational restrictions imposed by the c-tetrasubstituted -amino residues . these results demonstrate that the i + 1 and i + 2 aib residues play a role in controlling the e - selective rcm behavior with catalysts 22 and 23 and this conformational bias can not be overcome by z - selective catalysts . these studies highlight the importance of peptide sequence and conformation in controlling olefin geometry in the macrocyclic product and point to further strategies aimed at understanding how these parameters affect the efficiency of z - selective rcm . in summary , we have reported the first examples of z - selective metathesis on peptides using cyclometalated ruthenium catalysts . by examining a broad range of canonical and noncanonical amino acids in cross metathesis , homodimerization , and ring - closing metathesis , we have gleaned important criteria for achieving high conversion while maintaining excellent z selectivity . the following insights based on our results are summarized : the side - chain identity of an amino acid can dictate the activities of catalysts 1 and 2 in cross metathesis and homodimerization . in general , amino acids bearing aliphatic or aromatic side chains ( e.g. , alanine , leucine , and phenylalanine ) are highly active in metathesis , with yields approaching 85% and 94% z selectivity . sterically hindered side chains ( e.g. , valine or isoleucine ) and amino acids bearing bulky protecting groups lead to lower conversions but without degradation of z selectivity . amino acids bearing carboxylate functionalities ( i.e. , glutamic acid and aspartic acid ) require protection , as substrates bearing acidic functionalities can lead to catalyst decomposition and diminished z selectivity . amino acids bearing thiols or thioethers generally deactivate cyclometalated ruthenium catalysts ; however , in some cases , the use of protecting groups can lead to productive turnovers . side chains bearing hydroxyl groups ( e.g. , serine or threonine ) are generally tolerated by catalysts 1 and 2 , whereas amino acids bearing heterocycles have variable activity . tryptophan is active in z - selective homodimerization and cross metathesis , whereas histidine is generally inactive . polar side chains bearing carboxamide ( i.e. , glutamine or asparagine ) or guanidinium ( i.e. , arginine ) functionalities are generally intolerant of cyclometalated ruthenium catalysts . protection of these side chains can restore catalyst activity , leading to products highly enriched in z - olefins . cross metathesis and homodimerzation of amino acids and peptides using z - selective ruthenium catalysts can be performed in a variety of solvents , provided that the acidity of the reaction medium is kept minimal . the use of solvents such as mecn , dmso , and dmf generally leads to lower conversions than the use of noncoordinating solvents ( e.g. , dce ) . the use of protic solvents ( e.g. , meoh , etoh , or h2o ) can lead to products enriched in z - olefins . prolonged reaction times in protic solvents can , in some cases , lead to decomposition of catalysts 1 and 2 . amino acids bearing allylic or homoallylic functionality are active in z - selective metathesis . in general , higher conversions in homodimerization and cm can be achieved using homoallylic functionalities , particularly for sterically hindered substrates . noncanonical amino acids containing allylic heteroatoms including those that could be incorporated into peptides and proteins are active in z - selective cross metathesis and follow trends similar to those of noncyclometalated ruthenium catalysts . the use of aqueous conditions in the presence of salts as additives appears to diminish the activities of catalysts 1 and 2 . cyclometalated ruthenium catalysts can be used to synthesize stapled peptides bearing hydrocarbon olefinic cross - links positioned at residues i , i + 4 or i , i + 7 . to probe the limits of these catalysts in peptide stapling , we exposed z - selective catalysts to aib - rich peptides bearing o - allyl serine cross - links positioned at residues i and i + 3 that predominantly give rise to highly e - selective macrocyclic products . in these cases , catalysts such as 1 failed to undergo z - selective rcm , suggesting that conformational restrictions imposed by substrates such as 20 can influence the activities of cyclometalated ruthenium catalysts 1 and 2 in rcm . notably , compounds 15 , 17 , and 19 represent the most complex substrates synthesized by cyclometalated ruthenium catalysts , and this bodes well for further studies aimed at applying z - selective metathesis on substrates bearing multiple functionalities . it is envisioned that the studies reported herein will serve as a guideline in choosing appropriate alkene cross partners or promoting rcm on peptides . we anticipate that cyclometalated ruthenium catalysts could access new structures and provide insight into the role of alkene geometry in the biological activities of stapled peptides . moreover , installation of z - alkenes into peptides and proteins could allow sites for further modifications . progress in the design and development of stereoselective ruthenium catalysts will continue to broaden the application of olefin metathesis in natural - product synthesis and in biology . ischemia - induced pathologic growth of new blood vessels and expansion of extracellular matrix ( ecm ) in association with the outgrowth of fibrovascular epiretinal membranes at the vitreoretinal interface is the pathological hallmark in proliferative diabetic retinopathy ( pdr ) and often leads to catastrophic loss of vision due to vitreous hemorrhage and/or traction retinal detachment . proliferative vitreoretinopathy ( pvr ) is a process of fibrocellular proliferation on either sides of the retina that may complicate rhegmatogenous retinal detachment . the formation and gradual contraction of epiretinal membranes causes a marked distortion of the retinal architecture and results in complex retinal detachments that are difficult to repair . angiogenesis , the growth of new vascular networks from preexisting ones , is under tight regulation by a dynamic balance between angiogenic stimulators and inhibitors . the biological process of fibrosis , typically associated with an abnormal accumulation of ecm , occurs in response to various stimuli in many biological systems . the key cellular mediator of fibrosis is the myofibroblast , a cell type differentiated from quiescent fibroblasts . these are contractile cells , characterized by the expression of -smooth muscle actin ( -sma ) , and their presence is a marker of progressive disease . previous studies have shown that -sma - expressing myofibroblasts are the principal cellular component of pdr and pvr epiretinal membranes [ 36 ] . inflammation , angiogenesis , and fibrosis are processes involved in the pathogenesis of proliferative vitreoretinal disorders , and the interplay between these events is under intense investigation [ 38 ] . a number of proinflammatory , proangiogenic , profibrogenic , and immunomodulating factors may be linked to the development and progression of proliferative vitreoretinal disorders , such as osteopontin ( opn ) , high - mobility group box-1 ( hmgb1 ) , connective tissue growth factor ( ctgf ) , and pigment epithelium - derived factor ( pedf ) . osteopontin is a phosphorylated acidic arginine - glycine - aspartate- ( rgd-)containing glycoprotein that exists both as an immobilized ecm component and as a soluble , multifunctional , proinflammatory cytokine that plays important roles in promoting inflammation [ 9 , 10 ] , tissue remodeling , fibrosis [ 9 , 1114 ] , and angiogenesis [ 1518 ] . many of these effects are mediated by the binding of opn to cd44 receptors and the surface integrin receptor v3 [ 15 , 16 , 19 ] . hmgb1 is a nonhistone dna - binding nuclear protein that is highly conserved during evolution . necrotic cell death can result in passive leakage of hmgb1 from the cell as the protein is then no longer bound to dna . in addition , hmgb1 can be actively secreted by different cell types , including activated monocytes and macrophages , mature dendritic cells , natural killer cells , and endothelial cells . extracellular hmgb1 functions as a proinflammatory cytokine [ 2023 ] and exhibits angiogenic [ 2427 ] and fibrogenic [ 2831 ] effects . ctgf is a cysteine - rich secretory protein that functions as a downstream mediator of transforming growth factor- action on connective tissue cells . it acts as a fibroblast chemoattractant and mitogen and also stimulates the production of ecm components in various fibroblast cultures [ 3234 ] . in addition , in vitro and in vivo studies demonstrated that ctgf exhibits angiogenic activities [ 35 , 36 ] . pedf is a 50 kda secreted glycoprotein that belongs to the noninhibitory serpin family group . pedf has been described as a natural inhibitor of both physiological and pathological angiogeneses with antioxidant , and anti - inflammatory effects . it has been demonstrated to function as a potent and broadly acting neurotrophic and neuroprotective factor that induces cell differentiation and protects neurons in the brain , eye , and spinal cord against a wide range of neurodegenerative insults [ 37 , 38 ] . in addition , pedf was recently shown to have antifibrogenic activity . to address mechanisms involved in the pathogenesis of proliferative vitreoretinal disorders and to identify molecular targets for treatment and/or preventive intervention , we measured the levels of opn , hmgb1 , ctgf , and pedf in the vitreous fluid from patients with pdr , pvr , and rhegmatogenous retinal detachment with no pvr ( rd ) . undiluted vitreous fluid samples ( 0.30.6 ml ) were obtained from 48 patients with pdr , 17 patients with pvr , and 30 patients with rd during pars plana vitrectomy . the indications for vitrectomy in patients with pdr were traction retinal detachment and/or nonclearing vitreous hemorrhage . in patients with pdr , the severity of retinal neovascular activity was graded clinically at the time of vitrectomy using previously published criteria . neovascularization was considered active if there were visible perfused new vessels on the retina or optic disc present within tractional epiretinal membranes . neovascularization was considered inactive ( involuted ) if only nonvascularized , white fibrotic epiretinal membranes were present . active pdr was present in 28 patients , and inactive pdr was present in 20 patients . vitreous samples were collected undiluted by manual suction into a syringe through the aspiration line of vitrectomy , before opening the infusion line . the samples were centrifuged ( 500 rpm for 10 min , 4c ) , and the supernatants were aliquoted and frozen at 80c until assay . the study was conducted according to the tenets of the declaration of helsinki , and informed consent was obtained from all patients . the study was approved by the research centre , college of medicine , king saud university . enzyme - linked immunosorbent assay ( elisa ) kit for human opn ( human osteopontin , duoset , cat no . an elisa kit for hmgb1 ( human high - mobility group box-1 , cat no . elisa kits for human ctgf ( human connective tissue growth factor , cat no : e0010h ) and human pedf ( human pigment epithelium - derived factor , cat no . csb - e08818h ) were purchased from uscn life science & tech co. , ltd . and cusabio biotech co. , ltd . the minimum detection limit of each elisa kit for opn , hmgb-1 , ctgf , and pedf are 2.5 , 200 , 15.6 , and 40 picograms / ml ( pg / ml ) , respectively . the elisa plate readings were done using fluostar omega - miroplate reader from bmg labtech , offenburg , germany . the quantification of human opn , hmgb-1 , ctgf and pedf in the vitreous fluid was determined using elisa kits according to the manufacturer 's instruction . for each elisa kit , the undiluted standard serves as the highest standard and calibrator diluents serve as the blank . depending upon the detection range for each elisa kit and the expression level of the particular molecule , vitreous samples were either directly used or diluted with calibrator diluents supplied with elisa kit . for the measurement of opn , 100 l of 1000-fold diluted vitreous samples were added into each of the elisa plate for the analysis . for the quantification of hmgb1 , 60 l of diluent buffer was added to each well of microtiter plate followed by the addition of 40 l of standard , positive control , and vitreous fluid . for the measurement of ctgf , and pedf , 100 l of 3-fold and 6-fold diluted vitreous were used , respectively , for elisa assay . as instructed in the kit manual , vitreous samples were incubated into each well of elisa plates . antibodies against opn , hmgb1 , ctgf and pedf conjugated to horseradish peroxidase were added to each well of the elisa plate . the reaction was stopped by the addition of 2n sulfuric acid , and optical density was read at 450 nm in microplate reader . each assay was performed in duplicate . using the 4-parameter fit logistic ( 4-pl ) curve equation , the concentration for each sample was calculated after multiplying with the dilution factors to get the actual reading for each sample . because of the large variances that we had in our data , we used the nonparametric mann - whitney test to compare means from two independent groups , and the nonparametric kruskal - wallis test was used for conducting analysis of variance ( anova ) to compare means from more than two independent groups . post - anova pairwise comparisons of means were conducted using the kruskal - wallis test . for three groups , exploratory logistic regression analysis , involving forcing entry into a logistic model the variables of interest , was conducted to identify the angiogenic and fibrogenic factors that had a significant association with the studied diseases . the mean level of each variable was used as the cut - off value for high versus low levels . spss version 15 and programs lr and 3s from bio - medical data processing version 2007 ( bmdp 2007 ) statistical software ( cork technology pack , model farm road , cord , ireland ) were used for the statistical analyses . opn , hmgb1 , and pedf were detected in all vitreous samples from patients with rd , pvr , and pdr . ctgf was detected in all vitreous samples from patients with pdr and pvr and in 11 ( 36.6% ) samples from rd patients . mean levels of opn , hmgb1 , ctgf , and pedf in vitreous samples from pdr patients were significantly higher than those in rd patients ( p < 0.001 ; p = 0.002 ; p < 0.001 ; p < 0.001 , resp . ; mean levels of ctgf and pedf in vitreous samples from pvr patients were significantly higher than those in rd patients ( p < 0.001 ; p = 0.004 , resp . ; mean levels of opn and hmgb1 from pvr patients were higher than those in rd patients , but the differences between the means were not statistically significant ( p = 0.425 ; p = 0.571 , resp . ; mann - whitney test ) ( table 1 ) . comparison of mean levels of angiogenesis and fibrogenesis regulatory factors among active pdr patients , inactive pdr patients , and rd patients was conducted using the kruskal - wallis test , and the results are shown in table 2 . mean levels differed significantly between the 3 groups for opn ( p < 0.001 ) , hmgb1 ( p = 0.002 ) , ctgf ( p < 0.001 ) , and pedf ( p < 0.001 ) . post - anova pairwise comparisons of means indicated that the mean opn level was significantly higher in patients with active pdr than in rd patients ( z = 4.11 ) . for hmgb1 , the mean levels were significantly higher in patients with active pdr and patients with inactive pdr than in rd patients ( z = 2.92 ; z = 2.97 , resp . ) . for ctgf , the mean levels were significantly higher in patients with active pdr and patients with inactive pdr than those in rd patients ( z = 6.24 ; z = 4.2 , resp . ) . for pedf , the mean levels were significantly higher in patients with active pdr and patients with inactive pdr than in rd patients ( z = 6.89 ; z = 3.59 , resp . ) . in addition , mean pedf level was significantly higher in patients with active pdr than in patients with inactive pdr ( z = 2.57 ) . when patients with pdr were divided into those with or without traction retinal detachment , the mean levels of angiogenesis and fibrogenesis regulatory factors differed significantly between pdr patients with traction retinal detachment , pdr patients without traction retinal detachment , and rd patients for opn ( p = 0.002 ) , hmgb1 ( p = 0.003 ) , ctgf ( p < 0.001 ) , and pedf ( p post - anova pairwise comparisons of means indicated that , for opn , the mean levels in pdr patients with or without traction retinal detachment were significantly higher than those in rd patients ( z = 5.18 ; z = 5.64 , resp . ) . for hmgb1 , the mean levels in pdr patients with or without traction retinal detachment were significantly higher than those for rd patients ( z = 2.53 ; z = 3.26 , resp . ) . for ctgf , the mean levels in pdr patients with or without traction retinal detachment were significantly higher than those in rd patients ( z = 4.72 ; z = 5.87 , resp . ) . for pedf , the mean levels in pdr patients with or without traction retinal detachment were significantly higher than those in rd patients ( z = 5.17 ; z = 5.62 , resp . ) . pdr was significantly associated with high levels of hmgb1 ( odds ratio = 7.39 ; 95% confidence interval = 2.1125.9 ) , ctgf ( odds ratio = 11.4 ; 95% confidence interval = 2.8745.3 ) , and pedf ( odds ratio = 7.70 ; 95% confidence interval = 1.7733.5 ) . active pdr was significantly associated with high levels of ctgf ( odds ratio = 8.29 ; 95% confidence interval = 2.4428.1 ) and pedf ( odds ratio = 7.66 ; 95% confidence interval = 2.0029.4 ) . pdr with traction retinal detachment was significantly associated with high levels of hmgb1 ( odds ratio = 5.07 ; 95% confidence interval = 1.3618.9 ) . none of the studied angiogenesis and fibrogenesis regulatory factors was significantly associated with pvr . in patients with pdr , there were significant correlations between the vitreous fluid levels of pedf and the levels of opn ( r = 0.544 , p = 0.0011 ) , hgmb1 ( r = 0.719 , p < 0.001 ) , and ctgf ( r = 0.715 , p < 0.001 ) . in addition , there were significant correlations between the vitreous fluid levels of ctgf and the levels of opn ( r = 0.490 , p = 0.002 ) and hmgb1 ( r = 0.369 , p = 0.027 ) ( table 4 ) . in patients with pvr , there were significant correlations between the vitreous fluid levels of opn and the levels of hgmb1 ( r = 0.484 , p = 0.049 ) and pedf ( r = 0.559 , p = 0.020 ) ( table 4 ) . in this study , we examined the levels of the angiogenic and fibrogenic factors opn , hmgb1 , and ctgf and the antiangiogenic and antifibrogenic pedf in the vitreous fluid from patients with pdr , pvr , and rd and their relationship with pdr clinical disease activity . we found upregulation of opn , hmgb1 , ctgf , and pedf in the vitreous from pdr patients with active neovascularization compared with patients with quiescent pdr , pvr , and rd . exploratory logistic regression analysis identified a significant association between pdr and high levels of hmgb1 , ctgf , and pedf , between active pdr and high levels of ctgf and pedf , and between pdr with traction retinal detachment and high levels of hmgb1 . furthermore , there were significant correlations between the levels of pedf and the levels of opn , hmgb1 , and ctgf in patients with pdr and between the levels of opn and the levels of hmgb1 and pedf in patients with pvr . in the present study , we report that opn was significantly upregulated in the vitreous fluid from patients with pdr and that opn levels were nonsignificantly elevated in the vitreous fluid from patients with pvr . in a previous study , kase et al . demonstrated increased levels of opn in the vitreous fluid from patients with diabetic retinopathy ; however , they studied only 11 cases . our subgroup analysis showed that opn levels in vitreous samples from active pdr cases were higher than those in quiescent cases . these results are in agreement with a previous report in which we demonstrated that opn was expressed by vascular endothelial cells and stromal cells in pdr fibrovascular epiretinal membranes and by -sma - expressing myofibroblasts in pvr epiretinal membranes and that there was a significant correlation between the level of vascularization in pdr epiretinal membranes and the expression of opn . taken together , these findings suggest a role for opn in the progression of pdr . in vitro and in vivo studies demonstrated that opn is an important angiogenic factor [ 1518 ] . in addition , opn is required for the activation , migration , proliferation , and differentiation of fibroblasts into -sma - expressing myofibroblasts [ 1113 ] and is upregulated in several fibrotic diseases [ 9 , 11 , 12 , 14 ] . our results are consistent with previous reports showing that the proinflammatory cytokine opn plays a role in the development of diabetic vascular complications [ 9 , 4345 ] . the proinflammatory cytokine hmgb1 [ 2023 ] exhibits angiogenic [ 2427 ] and fibrogenic [ 2831 ] effects . another interesting role of hmgb1 in neovascularization is its ability to attract endothelial progenitor cells to sites of tissue injury and tumors to improve neovascularization . several studies showed overexpression of hmgb1 in other fibrotic disorders [ 28 , 29 , 31 ] . in vitro studies demonstrated that hmgb1 stimulated the proliferation and migration of fibroblasts [ 28 , 30 ] . in addition , exposure of epithelial cells to hmgb1 resulted in the transition from an epithelial to myofibroblast - like phenotype , with a significant increase in the mesenchymal markers -sma and vimentin . recently , arimura et al . demonstrated that hmgb1 stimulated the migration of human retinal pigment epithelial cells . in the present study , we report that hmgb1 was significantly upregulated in the vitreous fluid from patients with pdr , particularly in patients with active neovascularization in agreement with our previous report . furthermore , exploratory logistic regression analysis demonstrated significant associations between high levels of hmgb1 and all pdr and pdr with traction retinal detachment . in addition , in this study , we demonstrated that pvr eyes had a 3-fold increase in the vitreous level of hmgb1 when compared with those with rd . these results are in agreement with a previous report in which we demonstrated that hmgb1 was expressed by -sma - positive myofibroblasts in pvr epiretinal membranes . in addition to its role in mediating inflammation , angiogenesis , and fibrogenesis , several studies demonstrated that extracellular hmgb1 can aggravate tissue damage in neuronal tissue after ischemia [ 4850 ] . recently , various studies suggested an important role for the proangiogenic [ 35 , 36 ] and profibrotic [ 3234 , 51 ] ctgf in the development of chronic diabetes - related end - organ complications , including diabetic nephropathy . in the present study , ctgf levels in the vitreous fluid from patients with pdr and pvr our observations are consistent with previous reports showing increased ctgf levels in the vitreous fluid from patients with pdr [ 5355 ] and pvr . however , levels of ctgf in the vitreous fluid from patients with pdr were 3-fold higher than those in patients with pvr and levels of ctgf were particularly high in pdr patients with active neovascularization . in addition , exploratory logistic regression analysis demonstrated significant associations between high levels of ctgf and all pdr and pdr with active neovascularization . our results are not in agreement with a previous report , in which kuiper et al . showed that ctgf levels in the vitreous fluid from patients with pdr were significantly associated with the degree of fibrosis . our results are in agreement with a previous report in which we demonstrated increased expression of ctgf in the retinas from subjects with diabetes and that ctgf was expressed by vascular endothelial cells and -sma - expressing myofibroblasts in pdr epiretinal membranes and by myofibroblasts in pvr epiretinal membranes . in addition , there was a significant correlation between the level of vascularization in pdr epiretinal membranes and the expression of ctgf . the activity of pedf is selective in that it targets only new vessel growth and spares the preexisting vasculature [ 37 , 38 , 56 ] . the results of different studies on pedf expression in the vitreous from patients with pdr are conflicting . several studies found significant decrease in vitreous pedf levels in patients with pdr [ 5759 ] . other studies found that pedf levels in patients with pdr were not different from those in the controls [ 60 , 61 ] . however , duh and associates demonstrated significant increase of pedf levels in the vitreous from patients with active pdr . we do not have an explanation for the differences ; however , differences maybe method related . in the present study , the levels of pedf in the vitreous from patients with pdr and pvr were significantly greater than those in patients with rd . in addition , pedf levels in the vitreous from patients with pdr were higher than those in patients with pvr . our subgroup analysis showed that pedf levels were significantly higher in the vitreous from patients with active pdr compared with patients with quiescent pdr . furthermore , exploratory logistic regression analysis demonstrated significant associations between high levels of pedf and all pdr and active pdr . similarly , studies of other angiogenic eye diseases demonstrated increased levels of pedf in the aqueous humor in patients with choroidal neovascularization and macular edema secondary to branch retinal vein occlusion . in the present study , we demonstrated that the vitreous fluids from patients with pdr and pvr express different regulators of angiogenesis and fibrogenesis . recently , lenga et al . showed that opn is required for the presence of hmgb1 in the focal adhesions of fibroblasts and for ctgf expression by fibroblasts in response to the proinflammatory cytokine transforming growth factor-1 . these findings suggest that hmgb1 , and ctgf serve to mediate the immune response attributed to opn and that the interaction between opn , hmgb1 , and ctgf modulates fibroblast functions . the significant positive correlations between pedf levels and the levels of opn , hmgb1 , and ctgf in the vitreous from patients with pdr in the present study echoed the findings of matsuoka et al . that both pedf and the angiogenic vegf have been strongly expressed temporally and spatially in the retina of diabetic rats . similarly , there was a significant positive correlation between the expression of vegf and pedf in patients with choroidal neovascularization . our findings suggest that increased levels of pedf in the vitreous of patients with pvr and pdr , particularly active pdr , may be a response to counteract the activity of the angiogenic and fibrogenic factors . in addition , our data suggest that a positive regulatory feedback loop may exist in pdr , such that increased opn , hmgb1 , and ctgf induced synthesis of pedf . in conclusion , our data suggest that the upregulation of opn , hmgb1 and ctgf contribute to the pathogenesis of proliferative vitreoretinal disorders and that increased levels of pedf may counteract the activity of angiogenic and fibrogenic factors during the progression of pdr and pvr . the opn / hmgb1/ctgf pathway maybe a novel therapeutic target to inhibit progression of pdr and pvr .

olefin metathesis has emerged as a promising strategy for modulating the stability and activity of biologically relevant compounds ; however , the ability to control olefin geometry in the product remains a challenge . recent advances in the design of cyclometalated ruthenium catalysts has led to new strategies for achieving such control with high fidelity and z selectivity , but the scope and limitations of these catalysts on substrates bearing multiple functionalities , including peptides , remained unexplored . herein , we report an assessment of various factors that contribute to both productive and nonproductive z - selective metathesis on peptides . the influence of sterics , side - chain identity , and preorganization through peptide secondary structure are explored by homodimerization , cross metathesis , and ring - closing metathesis . our results indicate that the amino acid side chain and identity of the olefin profoundly influence the activity of cyclometalated ruthenium catalysts in z - selective metathesis . the criteria set forth for achieving high conversion and z selectivity are highlighted by cross metathesis and ring - closing metathesis on diverse peptide substrates . the principles outlined in this report are important not only for expanding the scope of z - selective olefin metathesis to peptides but also for applying stereoselective olefin metathesis in general synthetic endeavors . the aim of this study was to determine the levels of the angiogenic and fibrogenic factors osteopontin ( opn ) , high - mobility group box-1 ( hmgb1 ) , and connective tissue growth factor ( ctgf ) and the antiangiogenic and antifibrogenic pigment epithelium - derived factor ( pedf ) in the vitreous fluid from patients with proliferative diabetic retinopathy ( pdr ) , proliferative vitreoretinopathy ( pvr ) , and rhegmatogenous retinal detachment with no pvr ( rd ) . vitreous samples from 48 pdr , 17 pvr and 30 rd patients were studied by enzyme - linked immunosorbent assay . opn , hmgb1 , ctgf , and pedf levels were significantly higher in pdr patients than in rd patients ( p < 0.001 ; 0.002 ; < 0.001 ; < 0.001 , resp . ) . ctgf and pedf levels were significantly higher in pvr patients than in rd patients ( p < 0.001 ; 0.004 , resp . ) . exploratory logistic regression analysis identified significant associations between pdr and high levels of hmgb1 , ctgf and pedf , between pdr with active neovascularization and high levels of ctgf and pedf , and between pdr with traction retinal detachment and high levels of hmgb1 . in patients with pdr , there were significant correlations between the levels of pedf and the levels of opn ( r = 0.544 , p = 0.001 ) , hmgb1 ( r = 0.719 , p < 0.001 ) , and ctgf ( r = 0.715 , p < 0.001 ) . in patients with pvr , there were significant correlations between the levels of opn and the levels of hmgb1 ( r = 0.484 , p = 0.049 ) and pedf ( r = 0.559 , p = 0.02 ) . our findings suggest that opn , hmgb1 , and ctgf contribute to the pathogenesis of proliferative vitreoretinal disorders and that increased levels of pedf may be a response to counterbalance the activity of angiogenic and fibrogenic factors in pdr and pvr .

patients who are edentulous in the lower jaw and have to wear lower denture would usually complain of the poor retention . the retention is directly related to the vertical and torsion forces received , in other words , denture resistance against separation force from its sit . denture stability is believed widely to be related to resistance against other forces like oblique and anterior - posterior forces . the patient s satisfaction is directly influenced by the amount of denture retention as it has been shown through several studies [ 24 ] . the need for correcting the patient s problems with faulty denture is an inevitable consequence of retention failure and residual ridge resorption . several different strategies have been introduced to overcome the problem , one of which is the use of dental implants . three distinct methods of prosthetic treatments based on implants in the lower jaw are : implant supported fixed prosthesis ( fp-1 , fp-2 , fp-3)removable implant supported overdentures ( rp-4)combined implant - tissue supported over - dentures ( rp-5 ) . implant supported fixed prosthesis ( fp-1 , fp-2 , fp-3 ) removable implant supported overdentures ( rp-4 ) combined implant - tissue supported over - dentures ( rp-5 ) . the third of the above mentioned methods has the advantage of reducing the number of implants with a more simplified prosthetic approach . intra - osseous implants are considered as acceptable abutments in conjunction to attachments for efficient retention of the overdentures . factors involved in choosing the type of attachment include available space , maintenance necessity , spare part availability and ease of change , force distribution to the soft tissue or implant and the level of retention . another important fact is the level of wear seen in attachments following the use of overdenture . the aim of this research was to compare the effects of three different attachments ; nobel biocare ball ( nbb ) , nobel biocare bar and clip ( nbbc ) and sterngold era red ( erar ) on the retention and resistance of implant - supported overdentures . this in vitro investigation was performed on a total of 15 samples divided into three groups of five . each group was designed to include the attachments with erar ( 0 degree era micro np abutment only , 1 mm cuff and micro era male - red overdenture 5 , sterngold , usa ) , the attachments with nbbc ( gold adapt & gold abutment bar and clip attachment , nobel biocare , sweden ) and attachments with nbb ( ball abutment titanium , branemark system , rp 1 mm and plastic cap , nobel biocare , sweden ) . all samples were tested for the retention and resistance of overdenture on vertical , oblique and anterior - posterior tensile force load . recordings were made after the first insertion of the overdenture ( sit . 1 ) and also following 100 times of insertion / removal process ( sit . a pair of implants , 3.7510 mm , ( mk iii , branemark system , nobel biocare , sweden ) was inserted at both sides of the midline at the symphysis region in a parallel position . a milling machine ( paraskop m , bremen , germany ) was employed to avoid any fault in the angulation of insertion ( fig 1-a ) . a framework of cobalt chromium alloy ( wiironit , bego , bremen , germany ) was fabricated to fit on the two implants and the model . metallic structure was used for its durability and stability in all stages of the experiment . four stainless steel bands were soldered to the framework , which was positioned at the anterior part of the overdenture close to the implants . the others were established on the midline in the buccal and lingual side of the ridge . the overdenture was attached to the testing machine through the hooks ( fig 1-b ) . a prototype of overdenture housing was then constructed over the metallic overdenture by use of a light cure acrylic resin ( vlmc , vita , badsackingen , germany ) ( fig 1-c ) . all overdenture housings were duplicated from the prototype and fabricated by use of a self cure acrylic resin ( orthocryl , dentaurum , ispringen , germany ) ( fig 1-d ) . therefore , an acrylic overdenture housing was made for each sample ( totally 15 ) . one part of the attachment was attached into the overdenture housing and the other part , the abutment , was screwed on the implant ( fig 1-e ) . after the confirmation of appropriate sitting of the overdenture and adaptation of attachments , a series of measurements were started for each sample . recordings were made at 51 mm / min speed similar to that of denture move at bite and mastication . vertical force was measured to assess the retention while oblique and anterior - posterior forces were evaluated for resistance testing . maximum dislodging force ( mdf ) was recorded at the point of complete detachment of the overdenture from the model . data were finally subjected to an analysis using one - way anova test as well as tukey s test . based on the results derived from the collected data , the clinical behavior of three different attachments tested were not similar during application of different forces . as the data was normally distributed , one - way anova test was performed to compare the differences among three attachments for their force tolerance . at the beginning ( sit . 1 ) , tukey s test revealed that there was significant differences between erar / nbb , erar / nbbc and nbbc / nbb for vertical and oblique tensile strengths ( p<0.001 ) . this result was reemphasized by one - way anova test ( p<0.001 ) . therefore , erar had the highest retention and oblique resistance and nbb had the least . for anterior posterior tensile strength , tukey s test showed significant differences between erar / nbbc and erar / nbb ( p<0.001 ) , while there was no significant difference between nbbc and nbb ( p=0.528 ) . so erar had the highest anterior - posterior resistance but nbbc and nbb had the same . figs 24 show mdf measured for these attachments under vertical , oblique and anterior - posterior tensile forces in both situations ( sit . 1 and 2 ) . 2 , these data were statistically significant in their differences among different attachments for vertical and oblique tensile strengths , using one - way anova test ( p<0.001 ) . however , tukey s test on nbbc / nbb showed no statistically significant difference for vertical ( p=0.198 ) and oblique tensile strengths ( p=0.462 ) . erar had the highest retention and oblique resistance , while nbbc and nbb had the same . a further kruskal - wallis test performed on three groups in anterior - posterior tensile strength showed significant differences ( p<0.01 ) . the paired t - test performed to evaluate the condition of the three attachments in two different situations ( sit . 1 and sit . this indicated that the insertion / removal of overdenture for 100 times caused the differences between sit . 1 and sit . 2 in all three groups ( erar , nbbc and nbb ) . over the past three decades the use of implant - tissue supported overdenture has raised considerably among dentists . it is mainly related to the rise in patient awareness and availability of attachments to the dentist . several force directions are considered as applied in the mouth during function of the overdenture with retention being the most critical issue . two important points of view are considered on the retention of prosthesis , including the patient s feeling and the dentist s assessment . the latter which is measured during maximum load force application was surveyed in this research . 1 , erar has the highest resistance against vertical , oblique and anterior - posterior tensile forces . this means that overdenture has a higher retention and resistance potential with a significant difference from the other two attachments . this difference could be due to the design of erar as being intra - radicular . this is explained further by a female segment on the abutment of the attachment in addition to high and parallel abutment walls ( 5 mm length ) . the nbb showed the least mdf against vertical , oblique and anterior - posterior tensile forces . this is concluded to be due to the extra - radicular design in addition to free movement of the male component inside the female component . retention and resistance of nbb was in the least level while erar was at the most with significant differences ( p<0.001 ) . anterior - posterior resistance of erar was also at the highest level as mentioned before , while no differences were established between nbbc and nbb ( p=0.528 ) . petropoulos has reported a roughly similar result on nbb and nbbc characteristics while results reported on erar were different from the result achieved in this research . this could be due to the use of sterngold era of intra - radicular type which was not the case in the earlier mentioned study . 2 ) showed a considerable reduction in retention and resistance of overdenture on attachments possibly due to constant wear ( p<0.01 ) . this difference was statistically significant even in several insertion/ removal tests which the level of mdf against vertical , oblique and anterior - posterior forces was higher in erar than the others ( p<0.001 ) . in sit . 2 , there were no differences in retention and oblique resistance of nbb and nbbc ( p=0.198 , p=0.462 ) , but the anterior - posterior resistance of nbb was more than nbbc ( p<0.01 ) . it could also be explained by the material the attachments were made of , resulting in a higher resistance to wear in nbb compared to nbbc . based on the results of this research , clinical behaviors of these attachments ( erar , nbbc , nbb ) have been well identified . therefore , the dentist can choose an appropriate attachment in implant - tissue supported overdentures according to the clinical situation . it is recommended to use erar attachment when a high amount of retention and resistance is necessary . for example , for a patient who has severe residual ridge resorption or used to remove and insert the denture frequently , erar attachment should be applied . nbb and nbbc attachments would be the choice when there is low demand of retention and resistance for overdenture . insertion and removal of overdenture gradually causes loss of retention and resistance due to the attachment s wear . snapping hip , also known as coxa saltans , is characterized by an audible snapping sound or sensation felt when the hip is in motion , and may be associated with pain1 ) . of different types , external snapping hip is accompanied by popping noise when the posterior border of the iliotibial band slides over the greater trochanter as the hip is flexed or extended , and confirmed a positive ober 's test2 ) . although snapping hip is usually painless , this syndrome may be painful when greater trochanteric bursitis is associated . for most cases , conservative and medical treatment showed good treatment response . when symptoms persist despite different conservative treatment , surgical intervention is considered . surgical treatment options are n - plasty with trochanteric bursectomy , z - plasty and others . although these invasive methods are effective in improving pain and snapping sound , skin incision leaves a large scar and the trendelenburg gait may follow as a complication3,4,5,6,7,8,9,10 ) . ilizaliturri et al.5 ) obtained comparable clinical results compared to open surgical techniques by performing arthroscopic iliotibial band release in 10 patients ( 11 cases ) . however , no literature has been reported on evaluating clinical results of arthroscopy for snapping hip . therefore , this study aimed to indentify benefits of arthroscopic treatment by evaluating clinical results of iliotibial band release and gluteal sling release performed arthroscopically and comparing the results with those of previous studies . this study was performed retrospectively by including 7 snapping hip patients who underwent arthroscopic iliotibial band release and gluteal sling release by a single surgeon from september 2011 to june 2013 . subjects were 2 men and 5 women , and 3 patients had snapping hip on both sides . the mean age at the time of surgery was 35 years ( range , 25 - 49 years ) , and the mean follow - up was 19 months ( range , 12 - 33 months ) . the surgical intervention was done for patients who were refractory to conservative treatment for at least 3 months . the average time period from snapping sound recognition to arthroscopic treatment was 3 years ( range , 2 - 10 years ) . repetitive pain over the greater trochanter with snapping sound was detected in all cases , and snapping sound was externally visible and audible in most cases . some patients complained of symptoms of pseudosubluxation appearing as a subjective feeling of impending hip dislocation . two patients ( 4 cases ) were unable to adduct both knees in 90 of hip flexion or cross one leg over the other among patients with external snapping hip on both sides . this study excluded patients with sequelae of childhood hip disease , prior hip fractures , a history of prior hip surgery . this study also excluded patients with other hip diseases confirmed by preoperative radiographic and physical exams , and patients who underwent simultaneous arthroscopic treatment for intra - articular pathologies due to femoroacetabular impingement syndrome or other causes . two arthroscopic portals were made on the superior and inferior aspects of the greater trochanter . a trocar was inserted through a 6 mm incision made at 1 cm below to the vastus lateralis ridge , and positioned between the greater trochanter and iliotibial band to secure a space between them by gently moving the trocar . the space was maintained by injecting about 40 ml of saline solution , and the second portal was made at 1 cm above the proximal greater trochanter by inserting the arthroscope and viewing inside ( fig . , a parallel incision was made to orientation of the iliotibial band , and then the iliotibial band was incised vertically before and after isolating it . subsequently , a criss - cross pattern of incised line was made , and a diamond - shaped defect was created by gradually cutting the incised line to four directions using the shaver and high frequency ablator . the release was performed until snapping symptom disappeared through adduction movement and flexion - extension exercise of the hip intra - operatively . the findings of thickening and hyperemia in the greater trochanteric bursa were observed in all cases . additional gluteal sling release was performed in insertion of the gluteus maximus in 2 patients with hip adduction difficulty ( fig . 2 ) . to reduce loading imposed to the iliotibial band and alleviate pain during ambulation , patients began ambulating using crutches on the first postoperative day without limitation of weight bearing . the subjects used a continuous passive motion ( cpm ) device for the first month postoperatively to prevent soft tissue adhesion around the trochanteric area and restore early range of motion in the hip . the patients were instructed to use the cpm device consistently at home after being discharged on the second postoperative day . they were instructed to ambulate using crutches for a month , and restricted from active abduction exercise over the first 6 postoperative weeks . the subjects were allowed to begin active abduction exercise after the 6 weeks , and physical activities and exercise at preoperative levels . to evaluate the degree of clinical improvement and hip function , the visual analog scale ( vas ) and modified harris hip score ( mhhs ) were measured preoperatively and at the last follow - up . the presence of hip abduction weakness and ambulation abnormality was examined by performing the trendelenburg test on the sixth postoperative week and third postoperative month . in addition , the presence of iliotibial band tear and complications such as surgical site infection was checked postoperatively , and the recurrence of limping and snapping sound was evaluated at final follow - up . this study was performed retrospectively by including 7 snapping hip patients who underwent arthroscopic iliotibial band release and gluteal sling release by a single surgeon from september 2011 to june 2013 . subjects were 2 men and 5 women , and 3 patients had snapping hip on both sides . the mean age at the time of surgery was 35 years ( range , 25 - 49 years ) , and the mean follow - up was 19 months ( range , 12 - 33 months ) . the surgical intervention was done for patients who were refractory to conservative treatment for at least 3 months . the average time period from snapping sound recognition to arthroscopic treatment was 3 years ( range , 2 - 10 years ) . repetitive pain over the greater trochanter with snapping sound was detected in all cases , and snapping sound was externally visible and audible in most cases . some patients complained of symptoms of pseudosubluxation appearing as a subjective feeling of impending hip dislocation . two patients ( 4 cases ) were unable to adduct both knees in 90 of hip flexion or cross one leg over the other among patients with external snapping hip on both sides . this study excluded patients with sequelae of childhood hip disease , prior hip fractures , a history of prior hip surgery . this study also excluded patients with other hip diseases confirmed by preoperative radiographic and physical exams , and patients who underwent simultaneous arthroscopic treatment for intra - articular pathologies due to femoroacetabular impingement syndrome or other causes . two arthroscopic portals were made on the superior and inferior aspects of the greater trochanter . a trocar was inserted through a 6 mm incision made at 1 cm below to the vastus lateralis ridge , and positioned between the greater trochanter and iliotibial band to secure a space between them by gently moving the trocar . the space was maintained by injecting about 40 ml of saline solution , and the second portal was made at 1 cm above the proximal greater trochanter by inserting the arthroscope and viewing inside ( fig . , a parallel incision was made to orientation of the iliotibial band , and then the iliotibial band was incised vertically before and after isolating it . subsequently , a criss - cross pattern of incised line was made , and a diamond - shaped defect was created by gradually cutting the incised line to four directions using the shaver and high frequency ablator . the release was performed until snapping symptom disappeared through adduction movement and flexion - extension exercise of the hip intra - operatively . the findings of thickening and hyperemia in the greater trochanteric bursa were observed in all cases . additional gluteal sling release was performed in insertion of the gluteus maximus in 2 patients with hip adduction difficulty ( fig . to reduce loading imposed to the iliotibial band and alleviate pain during ambulation , patients began ambulating using crutches on the first postoperative day without limitation of weight bearing . the subjects used a continuous passive motion ( cpm ) device for the first month postoperatively to prevent soft tissue adhesion around the trochanteric area and restore early range of motion in the hip . the patients were instructed to use the cpm device consistently at home after being discharged on the second postoperative day . they were instructed to ambulate using crutches for a month , and restricted from active abduction exercise over the first 6 postoperative weeks . the subjects were allowed to begin active abduction exercise after the 6 weeks , and physical activities and exercise at preoperative levels . to evaluate the degree of clinical improvement and hip function , the visual analog scale ( vas ) and modified harris hip score ( mhhs ) were measured preoperatively and at the last follow - up . the presence of hip abduction weakness and ambulation abnormality was examined by performing the trendelenburg test on the sixth postoperative week and third postoperative month . in addition , the presence of iliotibial band tear and complications such as surgical site infection was checked postoperatively , and the recurrence of limping and snapping sound was evaluated at final follow - up . the vas decreased from 6.8 ( range , 6 - 9 ) preoperatively to 0.2 ( range , 0 - 2 ) postoperatively , and the mhhs improved from 68.2 ( range , 43 - 73 ) to 94.8 ( range , 89 - 100 ) after surgery . of 7 patients , 6 were pain - free at final follow - up , but a patient complained of mild pain at about vas score of 2 . hip adduction angles improved from 40 and 30 , respectively , to 0in two patients with adduction difficulty . moreover , they were able to cross one leg over the other in a sitting position . all patients had a negative trendelenburg 's test on the sixth postoperative week , and no patient had limping at the last follow - up . all patients were satisfied with scar aesthetically , and there was no recurrence of snapping sound . external snapping hip commonly occurs due to contracture and tension of the iliotibial band , which results in a positive ober 's test . the ober 's test is performed to identify tightness of the illiotibial band , and can be indicative for adduction difficulty when putting legs down suddenly after knee flexion at 90 and hip abduction in a lateral position . however , snapping sound can rarely occur due to contracture and tension of multiple fibrous bands of the gluteus maximus . in this case , although snapping hip is usually painless , this syndrome may be painful when greater trochanteric bursitis is associated . the greater trochanteric bursa is located between the greater trochanter , belonging to insertion of the gluteus medius and origin of the vastus lateralis , and iliotibial band , and pain can be generated from inflammatory response caused by snapping12 ) . most patients with snapping hip show good results with stretching exercise , antiinflammatory analgesic use , or greater trochanteric bursa injection3,4,5,8,13,14 ) . however , patients may be unable to sit cross - legged or adduct both legs in a sitting position due to pain like those with adduction difficulty in this study . when pain and discomfort persist despite conservative treatment , although a wide range of surgical techniques have been reported in previous literature , open surgical approaches have been most commonly introduced3,4,9,15,16 ) . after ilizaliturri et al.5 ) have addressed the result of arthroscopic treatment , successive results have been continuously reported in international studies1,8 ) . although surgical management of external snapping hip showed relatively favorable results , snapping or pain persisted postoperatively in some cases requiring reoperation3,4,5,6,7,15)(table 1 ) . with an average of 19-month follow - up after arthroscopic surgery , snapping symptom although a patient ( 1 case ) complained of mild pain at about vas score of 2 , the patient had no physical limitations in activities of daily living . although specific hip arthroscopic instruments should be available to treat intra - articular pathologies , knee or shoulder - length arthroscopic tools can be used to treat snapping hip . compared to an open z - plasty release , arthroscopy has a disadvantage of expensive cost . although not happened in this study , a defect which was created during iliotibial band release and not reconstructed posteoperatively had a risk of abduction weakness or depression on the surface of the thigh1 ) . limitations of this study were the relatively small sample size and short follow - up period and a retrospective study design . since snapping hip improves with conservative management in most patients , only a few patients are eligible to receive surgical treatment and it is difficult to include a large number of subjects7 ) . however , the long - term follow - up is anticipated to be favorable in identifying relapse of symptoms and the risk of complications in order to prove the effectiveness of arthroscopic intervention . another limitation was that this study did not measure hip abductor muscle strength between the affected and healthy sides before and after the surgery . since the iliotibial band acts as a static stabilizer of the hip , a diamond - shaped release of the iliotibial band weakens abductor muscle strength of the affected side . as a result , therefore , measuring abductor muscle strength of both sides pre- and post - operatively is thought to be helpful in analyzing the effect of iliotibial band release on changes in abductor muscle strength . however , this study was able to measure changes in muscle strength indirectly by regularly examining ambulation impairment caused by weakened abductor muscle strength by performing the trendelenburg test on the sixth postoperative week and third postoperative month . patients with external snapping hip recalcitrant to conservative management underwent arthroscopic treatment . despite a short mean follow - up of 19 months , we anticipate the successful treatment of patients with adduction difficulty due to contracture and tension of the gluteus maximus .

objective : retention and resistance of the implant - tissue supported overdenture may be affected by the type of attachment . the aim of this research was to compare the retention and resistance of nobel biocare ball ( nbb ) , nobel biocare bar and clip ( nbbc ) and sterngold era red ( erar ) attachments on an implant - tissue supported overdenture model.materials and methods : the attachment samples were divided into 3 groups of nbb , nbbc , and erar ( 5 samples in each group ) . two parallel nobel biocare branemark implants were placed symmetrically at the symphysis region of a mandibular test model . a metallic overdenture was fabricated precisely adapted to the model and attached to a zwick testing machine ( crosshead speed of 51 mm / min ) . dislodging tensile forces were applied in three vertical , oblique , anterior - posterior directions and two situations , at the beginning and after 100 times of insertion / removal of the overdenture , for each sample . the maximum dislodging force was measured . a one - way anova test was employed followed by tukey s test.results:erar was the most retentive and resistant in both situations . nbb and nbbc showed the same anterior - posterior resistance at the beginning . all test groups represented a large amount of retention and resistance loss after the insertion / removal of the overdenture , while nbbc showed a higher loss of anterior - posterior resistance than nb.conclusion:a highest level of retention and resistance was seen in erar . the retention and resistance were affected by the wear of attachments . purposethe purpose of this study was to evaluate the clinical outcome of arthroscopic treatment for recalcitrant external snapping hip.materials and methodsbetween september 2011 and june 2013 , we evaluated 7 patients ( 10 cases ) with snapping hip who were refractory to conservative treatments for at least 3 months . two patients ( 4 cases ) were impossible to adduct both knees in 90of hip flexion . surgery was done in lateral decubitus position , under spinal anesthesia . we made 2 arthroscopic portals to operate the patients , and used cross - cutting with flap resection technique to treat the lesion . we performed additional gluteal sling release in those 2 patients ( 4 cases ) with adduction difficulty . average follow - up length was 19 months ( range , 12 - 33 months ) . clinical improvement was evaluated with visual analog scale ( vas ) , modified harris hip score ( mhhs ) , and also investigated for presence of limping or other complications as well.resultsthe vas decreased from 6.8 ( range , 6 - 9 ) preoperatively to 0.2 ( range , 0 - 2 ) postoperatively , and the mhhs improved from 68.2 to 94.8 after surgery . none of the patients complained of post - operative wound problem or surgical complications.conclusionthe clinical outcome of arthroscopic treatment for recalcitrant external snapping hip was encouraging and all patients were also satisfied with the cosmetic results .

colchicine is a widely used drug for treatment of familial mediterranean fever ( fmf ) . clinical manifestations of colchicine intoxication include abdominal cramps , diarrhea , myotoxicity , hemolytic anemia and ( pan)cytopenia . a 9-years - old female patient , receiving colchicine for four months with a dose of 1 mg / day for fmf , was admitted to a hospital with gastrointestinal disturbance four days ago . she was referred to our hospital with liver and kidney dysfunction . at the initial physical examination her laboratory results revealed : hemoglobin 13.6 g / dl ; leukocyte , 9.610/l ; thrombocyte 5710/l ; fibrinogen 157 mg / dl ( 230 - 500 ) ; pt 26.1 sec ; inr 2.3 ; aptt 43.8 sec ; ldh 5329 iu / l ; ferritin 2320 g / l ( 10 - 55 ) ; triglyceride 7.1 mmol / l ( 0.32 - 1.46 ) . the patient was taken to intensive care unit ; vitamin k and fresh frozen plasma were administered . on the second day of her hospitalization hemoglobin was 9.6 g / dl ; leukocyte was 2.310/l ; neutrophil was 0.7210/l and thrombocyte was 2410/l . on peripheral blood smear , loss of lobulation in neutrophils was detected ( pelger - hut anomaly ) . laboratory tests revealed ferritin 54,632 g / l , triglyceride 7.4 mmol / l , and fibrinogen 63 mg / dl , serum creatinine 3.2 mg / dl ( 0.4 - 1.4 ) , serum blood urea nitrogen 43 mg / dl ( 6 - 21 ) , serum sodium 131 meq / l ( 134 - 148 ) , potassium 5.2 meq / l ( 3 - 4.8 ) , calcium 8.1 mg / dl ( 7.9 - 9.9 ) , and inorganic phosphate 2.1 mg / dl ( 2.4 - 4.7 ) . at the bone marrow aspiration , many pelger - hut cells were observed . soluble cd25 level was 2840 u / ml ( 220 - 710 ) , and creatine kinase level was 18,959 when the history of colchicine intoxication was detailed , during fmf attack - free period of five days prior to admission to the hospital , it is learned that the patient was upset over a quarrel between her parents , and that she had taken approximately 30 pills of her drug ( total of 15 mg ; 0.5 mg / kg body weight ) , at which vomiting and diarrhea complaints were started . the patient was administered hlh-2004 protocol and plasma exchange is applied twice . on the second day of therapy , severe hypotension , renal failure and av complete block occurred . the patient was resistant to all therapies and had died at the fourth day of her hospitalization . in the colchicine intoxication , the most affected organs are those that have a rapid cell turnover . liver damage , renal failure , arrhythmias , neuromuscular disturbances , and bone marrow depression can be detected in the later stage . the worst predicted doses were classified regarding the risk of toxicity as sub - toxic ( < 0.5 mg / kg ) , toxic ( 0.5 - 0.8 mg / kg ) , and lethal ( > 0.8 mg / kg ) doses . however , cases with cardiogenic shock and death were also reported with doses less than 0.5 mg / kg . although our patient had taken a dose of 0.5 mg / kg body weight colchicine , the patient died due to severe myelosupression , hlh and multi - organ failure . pelger - hut anomaly is a state of limitation in segmentation of lobes in neutrophils . a pelger - hut - like change in granulocyte morphology may occur as an acquired condition in several diseases . this same finding may be produced by certain drugs such as colchicine . on the peripheral blood smear of our patient , pelger - hut cells were detected in myeloid series , and as the history of colchicine use was detailed , it is learned that the patient had taken lots of colchicine pills . on the examination of concurrently taken bone marrow , pelger - hut cells were also detected in the myeloid series as well as hemophagocytosis . for this reason , we suggest that hlh was not due to her disease . to our knowledge , this is the first reported case of a patient with hlh associated with colchicine intoxication . in conclusion , if patients with colchicine intoxication present with pelger - hut anomaly in addition with unexplained fever , cytopenia , organomegaly , and biochemical changes , hlh should be investigated for prompt diagnosis and treatment . a hundred and fifty patients with end - stage renal disease were assessed for eligibility . the study was a prospective , randomized , controlled clinical trial ( iranian clinical registration number 138811182370n6 ) , and conducted at two tertiary - care urban medical center . patients with end - stage renal disease from december 2009 to march 2010 were included in the study . inclusion criteria included age more than 18 , being dialyzed with central tunneled catheter , med comp ( only if placed in the internal jugular vein ) , with a maximum time of one month post catheterization , dialysis 3 times a week and having accepted to participate in the study . exclusion criteria were allergy to cefotaxime , antibiotic treatment within 2 weeks prior to enrollment , patients requiring a surrogate decision maker , catheters with blood flow rates less than 300 ml / min , or requiring frequent thrombolytic solution dwells in the catheter lumen because of malfunction . at the beginning of the study 38 patients were eligible to participate in the study , but 30 of them were consented ( 20.6% ) . enrolled patients were randomly assigned by using a block computerized randomization protocol into two groups of 15 patients . in intervention group , we used cefotaxime , at a concentration of 10 mg / ml as the experimental antibiotic lock solution . solutions were prepared by dissolving sterile cefotaxime sodium powder in normal saline for injection to reach a concentration of 10 mg / ml for cefotaxime . then mixture of 1.5 cc cefotaxime ( 10 mg / ml ) and 1.5 cc heparin solution ( 5,000 u / ml ) ( ce / hs ) , was prepared in a syringe using aseptic precautions to fill 1.6 ml in the venous and 1.4 ml in the arterial lumen of the catheter at the end of each dialysis session ( exact volume of each venous and arterial port so that cefotaxime would not inter the blood stream , preventing its systemic effect and microbial resistance ) . in control group we just filled 1.6 ml and 1.4 ml of standard heparin solution ( hs ; 5,000 u / ml ) in the venous and arterial lumens respectively . in both groups , the catheter exit - site was washed with antiseptic solution at the initiation and termination of each session , and covered by topical mupirocin and dry sterile gauze during dialysis intervals . antibiotic solutions were stored in the refrigerator and nurses were trained to instill the solution into each of the two ports of the hd catheter at the end of each dialysis session , and withdraw it immediately before the next dialysis session . all the included patients received dialysis three times a week with the same catheter and the same exit site care , and they all had the same duration of catheter insertion . enrolled patients were routinely monitored clinically for symptoms and signs of infection , during each session of dialysis . blood cultures were drawn if patients had fever , chills , rigors , sweats , change in mental status , or exit - site infection4 . other causes of infection were ruled out . the study protocol required peripheral and catheter blood cultures and an exit site swab to be collected , if clinically indicated . if the catheter was removed , the catheter tip was cultured . patients with positive blood culture results were treated with systemic antibiotics driven by type of organism and antibiotic susceptibility . blood stream infections were defined by the cdc ( centers for disease control ) criteria21 . definite blood stream infection was defined as the same organism from a semiquantitative culture of the catheter tip ( 15 colony - forming units / catheter segments ) or catheter blood sample versus peripheral blood sample in a symptomatic patient with no other apparent source of infection22 . if colony count of catheter were 5 times more than blood sample , blood stream infection were established720 . . statistical analysis was performed with independent sample t - test and chi - square tests . a hundred and fifty patients with end - stage renal disease were assessed for eligibility . the study was a prospective , randomized , controlled clinical trial ( iranian clinical registration number 138811182370n6 ) , and conducted at two tertiary - care urban medical center . patients with end - stage renal disease from december 2009 to march 2010 were included in the study . inclusion criteria included age more than 18 , being dialyzed with central tunneled catheter , med comp ( only if placed in the internal jugular vein ) , with a maximum time of one month post catheterization , dialysis 3 times a week and having accepted to participate in the study . exclusion criteria were allergy to cefotaxime , antibiotic treatment within 2 weeks prior to enrollment , patients requiring a surrogate decision maker , catheters with blood flow rates less than 300 ml / min , or requiring frequent thrombolytic solution dwells in the catheter lumen because of malfunction . at the beginning of the study 38 patients were eligible to participate in the study , but 30 of them were consented ( 20.6% ) . enrolled patients were randomly assigned by using a block computerized randomization protocol into two groups of 15 patients . in intervention group , we used cefotaxime , at a concentration of 10 mg / ml as the experimental antibiotic lock solution . solutions were prepared by dissolving sterile cefotaxime sodium powder in normal saline for injection to reach a concentration of 10 mg / ml for cefotaxime . then mixture of 1.5 cc cefotaxime ( 10 mg / ml ) and 1.5 cc heparin solution ( 5,000 u / ml ) ( ce / hs ) , was prepared in a syringe using aseptic precautions to fill 1.6 ml in the venous and 1.4 ml in the arterial lumen of the catheter at the end of each dialysis session ( exact volume of each venous and arterial port so that cefotaxime would not inter the blood stream , preventing its systemic effect and microbial resistance ) . in control group we just filled 1.6 ml and 1.4 ml of standard heparin solution ( hs ; 5,000 u / ml ) in the venous and arterial lumens respectively . in both groups , the catheter exit - site was washed with antiseptic solution at the initiation and termination of each session , and covered by topical mupirocin and dry sterile gauze during dialysis intervals . antibiotic solutions were stored in the refrigerator and nurses were trained to instill the solution into each of the two ports of the hd catheter at the end of each dialysis session , and withdraw it immediately before the next dialysis session . all the included patients received dialysis three times a week with the same catheter and the same exit site care , and they all had the same duration of catheter insertion . enrolled patients were routinely monitored clinically for symptoms and signs of infection , during each session of dialysis . blood cultures were drawn if patients had fever , chills , rigors , sweats , change in mental status , or exit - site infection4 . other causes of infection were ruled out . the study protocol required peripheral and catheter blood cultures and an exit site swab to be collected , if clinically indicated . if the catheter was removed , the catheter tip was cultured . patients with positive blood culture results were treated with systemic antibiotics driven by type of organism and antibiotic susceptibility . blood stream infections were defined by the cdc ( centers for disease control ) criteria21 . definite blood stream infection was defined as the same organism from a semiquantitative culture of the catheter tip ( 15 colony - forming units / catheter segments ) or catheter blood sample versus peripheral blood sample in a symptomatic patient with no other apparent source of infection22 . if colony count of catheter were 5 times more than blood sample , blood stream infection were established720 . . statistical analysis was performed with independent sample t - test and chi - square tests . a hundred and fifty patients with end - stage renal disease were assessed for eligibility . the study was a prospective , randomized , controlled clinical trial ( iranian clinical registration number 138811182370n6 ) , and conducted at two tertiary - care urban medical center . patients with end - stage renal disease from december 2009 to march 2010 were included in the study . inclusion criteria included age more than 18 , being dialyzed with central tunneled catheter , med comp ( only if placed in the internal jugular vein ) , with a maximum time of one month post catheterization , dialysis 3 times a week and having accepted to participate in the study . exclusion criteria were allergy to cefotaxime , antibiotic treatment within 2 weeks prior to enrollment , patients requiring a surrogate decision maker , catheters with blood flow rates less than 300 ml / min , or requiring frequent thrombolytic solution dwells in the catheter lumen because of malfunction . at the beginning of the study 38 patients were eligible to participate in the study , but 30 of them were consented ( 20.6% ) . enrolled patients were randomly assigned by using a block computerized randomization protocol into two groups of 15 patients . in intervention group , we used cefotaxime , at a concentration of 10 mg / ml as the experimental antibiotic lock solution . solutions were prepared by dissolving sterile cefotaxime sodium powder in normal saline for injection to reach a concentration of 10 mg / ml for cefotaxime . then mixture of 1.5 cc cefotaxime ( 10 mg / ml ) and 1.5 cc heparin solution ( 5,000 u / ml ) ( ce / hs ) , was prepared in a syringe using aseptic precautions to fill 1.6 ml in the venous and 1.4 ml in the arterial lumen of the catheter at the end of each dialysis session ( exact volume of each venous and arterial port so that cefotaxime would not inter the blood stream , preventing its systemic effect and microbial resistance ) . in control group we just filled 1.6 ml and 1.4 ml of standard heparin solution ( hs ; 5,000 u / ml ) in the venous and arterial lumens respectively . in both groups , the catheter exit - site was washed with antiseptic solution at the initiation and termination of each session , and covered by topical mupirocin and dry sterile gauze during dialysis intervals . antibiotic solutions were stored in the refrigerator and nurses were trained to instill the solution into each of the two ports of the hd catheter at the end of each dialysis session , and withdraw it immediately before the next dialysis session . all the included patients received dialysis three times a week with the same catheter and the same exit site care , and they all had the same duration of catheter insertion . enrolled patients were routinely monitored clinically for symptoms and signs of infection , during each session of dialysis . blood cultures were drawn if patients had fever , chills , rigors , sweats , change in mental status , or exit - site infection4 . other causes of infection were ruled out . the study protocol required peripheral and catheter blood cultures and an exit site swab to be collected , if clinically indicated . if the catheter was removed , the catheter tip was cultured . patients with positive blood culture results were treated with systemic antibiotics driven by type of organism and antibiotic susceptibility . blood stream infections were defined by the cdc ( centers for disease control ) criteria21 . definite blood stream infection was defined as the same organism from a semiquantitative culture of the catheter tip ( 15 colony - forming units / catheter segments ) or catheter blood sample versus peripheral blood sample in a symptomatic patient with no other apparent source of infection22 . if colony count of catheter were 5 times more than blood sample , blood stream infection were established720 . data were analyzed using spss software ( spss inc . , chicago , il ; ver.18 ) . statistical analysis was performed with independent sample t - test and chi - square tests . all patients were visited in the hd unit on every other daily basis , and clinical events were closely examined . baseline demographic and clinical data including , age , male - female ratio , weight , diabetes , hypertension , lab data ( complete blood count , serum ferritin and serum albumin ) did not have significant difference among two groups ( p > 0.05 ) ( table 1 ) . demographic and basic clinical characteristics of patients at the beginning of study exit site infection was occurred in neither of groups but eleven patients developed cri in the hs ( control ) group at the time of the first interim analysis , whereas no patient had cri in the ce / hs group . clinical examination and follow - up of the patients with infection infection rate per 1000 catheter - days was zero in the ce / hs group versus 6.84 in the hs group ( p < 0.001 ) . the bacteria isolated from peripheral and catheter blood were gram - negative bacilli in 64% , while 36% of them were grampositive cocci . among gram - positive cocci , infection - free survival rates at 180 days were 100% for the ( ce / hs ) group , and 56% for the hs group ( p < 0.001 , pearson chi - square value = 17.368 ) . cri results from migration of skin organisms along the catheter into the bloodstream or contamination and colonization of catheter lumens.23 all indwelling vascular catheters develop a biofilm on internal and external surfaces . subsequent colonization of this biofilm occurs in a high percentage of catheters and precedes bacteremia and septic symptoms.24 through reduction in cri rate , we can reach dramatic reduction in morbidity and mortality of hemodialysis patients . antibiotic lock is one of the methods which can help to prevent cri , but according to its importance and financial cost , prospective randomized clinical trials are necessary to confirm its efficacy . the aim of this study was to determine the efficacy of catheter restricted filling with cefotaxime and heparin in preventing tunneled catheter related infections ( cri ) in hemodialysis patients . patients with confirmed bacteremia may benefit from administration of intravenous antibiotics and instillation of the antibiotic - heparin lock between dialysis sessions without catheter removal . a lower concentration of the antibiotic - heparin lock was investigated for preventing gram - positive central venous catheter related infections in 57 neutropenic patients with cancer.25 antiseptic and antibiotic coatings on central venous catheters showed promising results in intensive care populations.2627 a 79% reduction in the rate of catheter - related blood stream infections ( relative risk [ rr ] 0.21 , 95% ci 0.03 - 0.95 ) was reported with use of chlorhexidine - silver sulfadiazine - coated catheters . application of these results to patients receiving chronic hemodialysis is limited as catheters used in intensive care unit are in situ for approximately one week , versus hemodialysis catheters which may be in situ for several weeks or even months.26 cefotaxime is active against most gram - negative bacilli ( except pseudomonas ) and gram - positive cocci ( except enterococcus ) and is also active against many penicillin - resistant pneumococci . this study suggested that infection rates per 1000 catheter - days were significantly lower in the ce / hs group versus hs ( control ) group ( 0.000 vs. 6.84/1000 catheter days ) . saxena showed a lower cri incidence in the ce / hs group versus hs ( control ) group in non - tunneled hd catheters ( 1.65 vs. 3.13/1000 catheter days ) . in tunneled hd catheters , rate of cri is less than non - tunneled hd catheters.4 furthermore , he showed a lower cri incidence in the ce / hs group versus hs ( control ) group ( 1.67 vs. 3.60/1000 catheter days ) in tcc.28 cri rates were significantly lower with the use of antibiotic locking solution ( alss ) . pooled data from several studies show that cri is 7.72 ( 95% confidence interval , 5.1 to 10.3 ) times less likely with als than with heparin.29 six of these studies used heparin , 5000 u / ml , as the control group , while the study by kim et al26 used 1000 u / ml . antibiotics tested include gentamicin,223031 gentamicin / citrate combination,32 cefotaxime,4 minocycline,22 cefazolin/ gentamicin combination26 and chlorhexidine.33 in this study , the choice of cefotaxime was based on its broad spectrum effect on gramnegative organisms in addition to being most effective amongst third generation cephalosporins against staphylococcus aureus . cefotaxime was preferred over gentamicin for locking tcc , which can be potentially ototoxic , specially in elderly patients who often have preexisting hearing disabilities.283435 the present study shows that catheter - restricted filling with a solution containing cefotaxime and heparin may significantly reduce the incidence of catheter - related infection among end stage renal disease patients . cefotaxime as an antibiotic lock solution appears to be effective and safe ; although there is some concern about the allergy to its leaked solution from lumens . therefore we excluded the patients who had allergy to cefotaxime . in order to omit biases in our study , all the patients had the same frequency of manipulation , they all received dialysis three times a week with the same catheter and they all had the same duration of catheter insertion . with the use of antibiotic lock method for reduction in cri rate , we may reach dramatic reduction in morbidity and mortality of hemodialysis patients . since this study was a small sample size and short - term follow - up study , and according to its importance and financial cost associated , there is a need for additional studies with long - term follow - up on larger population to evaluate the efficacy of cefotaxime lock protocol and its probable adverse effects and its correlation with using topical agents . mm , sa , bp , and aa selected and managed patients , fs referred eligible patients after catheter placement , ma was our statistician , rs , ahdj , and ns were the major contributors in writing the manuscript .

colchicine is frequently used in the treatment of familial mediterranean fever ( fmf ) . first symptoms of colchicine intoxication are gastrointestinal disturbances , such as abdominal cramps , diarrhea , pancytopenia and so on . herein , we report a female fmf patient with pancytopenia and hemophagocytic lymphohitiocytosis ( hlh ) , following colchicine intoxication for committing suicide . to our knowledge , this is the first reported case of a patient with hlh associated with colchicine intoxication . background : chronic hemodialysis patients frequently require vascular access through central venous catheters ( cvcs ) . the most significant complication of these catheters is infection . this risk can be lowered by the use of an antibiotic - heparin lock . this study focuses on hemodialysis patients using tunneled - cuffed catheters ( tcc ) , to assess the rate of catheter - related infections ( cri ) in catheter - restricted filling with cefotaxime and heparin in end stage renal disease patients.methods:a double - blind randomized study was conducted to compare 5000 u / ml heparin plus10 mg / ml cefotaxime ( ce / hs ) as catheter - lock solutions , with heparin ( 5000 u / ml ) alone . a total of 30 patients with end - stage renal disease and different etiologies , were enrolled for chronic hemodialysis with permanent catheters from december 2009 to march 2010 . these patients were randomly assigned to two groups of 15 members . blood samples were collected for culture , sensitivity , and colony count , from the catheter lumen and the peripheral vein . cri was considered as the end point.results:the rate of cri was significantly lower in the cefotaxime group versus control group ( p < 0.001 ) . no exit site infection was occurred in both groups . infection - free survival rates at 180 days were 100% for the ce / hs group , and 56% for the hs group.conclusions:antibiotic lock therapy using cefotaxime reduces the risk of cri in hemodialysis patients .

mechanical plaque control is largely the responsibility of the individual using toothbrushes and interdental cleaning devices . despite one 's best efforts , mechanical aids fail to adequately remove plaque biofilm , for which chemical plaque control is often recommended as an adjunct to mechanical plaque control to help maintain gingival health . a number of chemical agents like phenolic compounds , bis - biguanides , pyrimidines , quaternary ammonium compounds , oxygenating agents , halogens , heavy metal salts which have antiseptic or antimicrobial action have been used , with variable success , to inhibit plaque formation and the development of gingivitis . chlorhexidine ( chx ) is the most studied and effective antiseptic for plaque inhibition and prevention of gingivitis when used twice daily as mouth rinse . chx besides its side effects including : brown discoloration of the teeth , some restorative materials and mucosa ; bitter taste and a slight increase in supragingival calculus formation is known as gold standard of antimicrobial rinses because of broad - spectrum activity and substantivity of 812 h. in order to overcome such side effects the world health organization advice researchers to investigate the possible use of natural products such as herb and plant extracts . a number of clinical studies have shown the effects of using mouthwashes extracted from herbs such as myrtus communis , qureucus infectoria , capparis spinosa , and cinnamon in the prevention of dental plaque accumulation and subsequent gingival inflammation . there is the minimum evidence that proves the efficacy of herbal mouth rinses and their ability to control plaque - induced gingivitis . thus , the aim of this study was to evaluate the clinical changes after the usage of herbal oral rinse and 0.12% chx . this randomized controlled clinical trial was carried out in the department of periodontology and implantology , m a rangoonwala college of dental sciences and research center , pune . the study population consisted of 152 individuals , who were systemically healthy , between 20 and 50 years of age and with moderate to severe plaque - induced gingivitis were enrolled in the study . they were equally distributed in the test ( n = 76 ) and the control group ( n = 76 ) . participants were excluded from the study if they suffered from nonplaque induced gingivitis or periodontitis , history of antibiotic use and use of any form of herbal products in the last 90 days , need for antibiotic premedication , patients using mouth rinse within the last 3 months , pregnant women , habit of smoking or any form of smokeless tobacco and with systemic diseases . patients were selected on the basis of inclusion and exclusion criteria and were randomly assigned using a coin toss to : group i ( test group ) : hiora herbal oral rinse that consists of piper betle ( nagavalli ) , bhibhitika ( terminalia bellerica ) , pilu ( salvadora persica ) commonly known as meswak , gandharpura tailum , yavani , ela , peppermint satvagroup ii ( control group ) : peridex that consists of 0.12% chx . group i ( test group ) : hiora herbal oral rinse that consists of piper betle ( nagavalli ) , bhibhitika ( terminalia bellerica ) , pilu ( salvadora persica ) commonly known as meswak , gandharpura tailum , yavani , ela , peppermint satva group ii ( control group ) : peridex that consists of 0.12% chx . the clinical examination included gingival index ( lo and silness , 1967 ) and plaque index ( silness and lo , 1964 ) which were recorded at baseline and postoperatively after 21 days . all the patients in group i and group ii were instructed to use the assigned mouth rinse 15 ml twice daily for 30 s in conjunction to their normal oral hygiene routine . participants from both the groups were advised to use the colgate medium bristle toothbrush and colgate total toothpaste . patients were recalled at weekly interval to check for the oral hygiene and the oral hygiene was reinforced in noncompliant patients . is done using independent sample t - test after confirming the underlying normality assumption . within group statistical comparison of all the parameters is done using paired sample t - test after confirming the underlying normality assumption of differences . the relative percentage change in both the parameters is calculated using following the formula : ( baseline - 21 days ) 100/(baseline ) . is done using independent sample t - test after confirming the underlying normality assumption . within group statistical comparison of all the parameters is done using paired sample t - test after confirming the underlying normality assumption of differences . the relative percentage change in both the parameters is calculated using following the formula : ( baseline - 21 days ) 100/(baseline ) . group i did not yield statistically significant results than group ii in the proportion of gingival index scores and plaque index scores of baseline parameters [ table 1 ] . intergroup comparison of baseline parameters when within group comparison of pretreatment and posttreatment scores was done , plaque index scores of posttreatment were statistically significant [ table 2 ] . when between group comparison of relative percentage change in study parameters was done group ii results were statistically significant as compared to group i [ table 3 ] . intragroup comparison of pre- and post - mouthwash treatment ( plaque index ) inter group comparison of relative percentage change in study parameters when within group comparison of pretreatment and posttreatment scores was done , gingival index scores of posttreatment were statistically significant [ table 4 ] . when between group comparison of relative percentage change in study parameters was done group ii results were statistically significant as compared to group i [ table 3 ] . when within group comparison of pretreatment and posttreatment scores was done , plaque index scores of posttreatment were statistically significant [ table 2 ] . when between group comparison of relative percentage change in study parameters was done group ii results were statistically significant as compared to group i [ table 3 ] . intragroup comparison of pre- and post - mouthwash treatment ( plaque index ) inter group comparison of relative percentage change in study parameters when within group comparison of pretreatment and posttreatment scores was done , gingival index scores of posttreatment were statistically significant [ table 4 ] . when between group comparison of relative percentage change in study parameters was done group ii results were statistically significant as compared to group i [ table 3 ] . the purpose of this study was to determine the comparative effects of herbal oral rinse ( hiora ) to 0.12% chx ( peridex ) on gingival health and plaque accumulation over time . hiora herbal oral rinse consists of p. betle ( nagavalli ) , bhibhitika ( t. bellerica ) , pilu ( s. persica ) commonly known as meswak , gandharpura tailum , yavani , ela , peppermint satva . suggests that there are certain ingredients in herbal oral rinses that exhibit evidence of anti - inflammatory and anti - fungal therapeutics effects . it is a medicinal plant that has been used by many people in africa , south america , middle east and asia . s. persica contains a number of identified antimicrobials and prophylactic components including fluorides , alkaloids , sulfur compounds and volatile oils such as benzyl isothiocyanate . they alter the characteristics of the early plaque settlers streptococcus sanguinis , streptococcus mitis and actinomyces species and make them less adherent . the anionic components s. persica has an antimicrobial activity against streptococcus aureus , streptococcus mutans , streptococcus fecalis , lactobacillus , pseudomonas aeruginosa , and candida albicans . in this regards , almas et al . compared antimicrobial activity of eight commercially available mouth rinses and 50% miswak extract against seven microorganisms . they found that mouth rinses containing chx had the maximum antibacterial activity while miswak extract had low antibacterial activity . hydrochavicol in p. betle inhibits expression of pro - inflammatory cytokine , tumor necrosis factor- , disrupts the permeability barrier of the microbial membrane of s. mutans and actinomyces species and also has an astringent action . methyl salicylate in gandharpura taila , cineole in ela , thymol in yavani and menthol in peppermint satva impart a fragrant and refreshing effect . additional research conducted by scherer et al . demonstrated that herbal oral rinse reduced gingival bleeding after 3 months of use as compared to placebo . chlorhexidine is effective against an array of microorganisms including gram - positive and gram - negative organisms , fungi , yeast and viruses . the ability of an oral rinse to be retained in the oral cavity and maintain potency over an extended length of time has been debated . lang stated the substantivity of an antimicrobial agent needs sufficient contact time with a microorganism in order to inhibit or kill it . chx , with a substantivity of 12 h is considered to be highly effective ; whereas , the substantivity of herbal mouth rinse is unknown . hence , in the present study , comparison was made between herbal oral rinse ( hiora ) and 0.12% chx ( peridex ) to see their effectiveness . in this study , there was a statistically significant reduction in the proportion of gingival index scores and plaque index scores in the chx group . however , in a study done by chatterjee et al . , 2011 herbal oral rinse is equally effective in reducing periodontal indices as chx . however , there is not enough statistically significant evidence to suggest that herbal oral rinse had a greater effect in reducing gingival index scores . with the proliferation of herbal oral care products , it is important for clinicians to make evidence - based decisions when making product recommendations . research needs to be conducted to determine the substantivity of herbal mouth rinse as well as to determine its antimicrobial effects on gingivitis , plaque biofilm accumulation , and related bacteria . suggestions for future studies include : ( 1 ) expand study population to include broader disease status , and varied age group ( 2 ) extend study to 6 months ( 3 ) add stains and calculus indices . within the limitations of the study when herbal oral rinse was compared to 0.12% chx , 0.12% chx mouth rinse effectively reduced the clinical symptoms of plaque - induced gingivitis , and had a statistically significant effect on the reduction of plaque scores . the contributing causes of dn pathogenesis and progression are still poorly understood but chronic hyperglycemia and high blood pressure represent the main risk factors for disease onset . , high systemic blood pressure usually determines an increase of the intraglomerular pressure and glomerular filtration rate ( gfr ) which results in glomerular hyperfiltration . from the biochemical point of view , hyperglycemia per se sustains the accumulation of advanced glycation end products ( ages ) , altering the electronegativity of the cell ; additionally ages bind proteins of the extracellular matrix ( ecm ) inhibiting their degradation . ages accumulation can induce an increased production of reactive oxygen species ( ros ) and a transcriptional activation of different proinflammatory and profibrotic molecules , including tgf - beta [ 2 , 3 ] . the high glucose - mediated induction of tgf - beta and the central role of this growth factor in dn progression represent the few defining constants in the pathogenesis of dn . the earliest clinical signs of dn include a slight but persistent urinary excretion of albumin ( microalbuminuria ) and a temporary increase of the glomerular filtration rate ( gfr ) . these clinical signs , along with the presence of hyperglycemia , are often considered sufficient indicators of dn [ 5 , 6 ] . today , extensive evidence shows that dn is not the only type of renal damage that can be found in diabetic patients [ 7 , 8 ] and kidney biopsy , although highly invasive , remains the diagnostic gold standard . the histological hallmarks of dn include hyperproliferation of the mesangial cells , thickening of the glomerular basement membrane ( gbm ) , podocyte effacement , tubulointerstitial fibrosis , and nodular accumulations of ecm ( kimmelstiel - wilson lesions ) in the glomerulus . given the high prevalence of type 2 diabetes ( t2d ) and the diagnostic limitations currently associated with kidney biopsy , there is an impending need for new , accurate , and easily accessible biomarkers of disease . in this review we will try to outline a system biology overview on dn by recapitulating the main annotations obtained at different levels of molecular investigation . only those studies investigating human samples will be described ; the murine models of dn in fact , although undergoing albuminuria , mesangial expansion , and podocyte loss , do not develop severe glomerulosclerosis and tubulointerstitial fibrosis . also , as substantial differences exist in the etiology and prevalence of type 1 and type 2 dn , the articles discussed in this paper apply to dn secondary to type 2 diabetes ( t2dn ) . as an exception , works describing biomarkers of kidney damage in t1d that have been further validated in t2 dm and vice versa and those reporting potential prognostic biomarkers , because of their particular importance in predicting the progression of renal damage , have been also discussed in the present work . all the annotations discussed in this review are also listed in tables 1 , 2 , 3 , 4 , and 5 , categorized according to whether they summarize the genetic and transcriptomic signature of coding or noncoding rna molecules and the epigenetic proteomic and metabolomic markers , respectively . genetic variation is present under different forms in the human genome , ranging from single nucleotide polymorphisms ( snps ) to large , structural , chromosomal rearrangements . today we know that genetic variation infers disease susceptibility and collective effort aims at identifying the precise loci for dn susceptibility . different methodological strategies can be used to characterize the genetic risk for a disease , either targeted or genome - wide , according to whether a priori hypothesis of the candidate regions for disease susceptibility exists . in genome - wide association studies ( gwas ) , for instance , the whole genome is screened for new , previously uncharacterized single nucleotide polymorphisms ( snps ) . prior to the development of the modern high - throughput technologies such as chip - based microarray analysis and next - generation sequencing , the inheritance of disease susceptibility was investigated through genetic linkage in families . basically , individuals within the same families were sequenced for a collection of genetic snps in order to identify those snps segregating with the disease . this approach led to the identification of many variants responsible for disease susceptibility but it proved mostly suitable for the study of single gene disorders . for complex , common complications like t2d in fact , progression is very likely driven by multiple alleles simultaneously , each having a small correlation to disease progression if inherited individually . this implies that a big population needs to be genotyped in order to detect the common variants responsible for the increased genetic risk . in the field of dn , there is extensive evidence for genetic contribution to disease susceptibility . in 1989 , seaquist et al . showed that diabetic siblings of patients with dn were more at risk for developing dn compared to diabetic siblings of diabetic patients without proteinuria ; epidemiologic studies also indicate that the prevalence of dn varies among ethnic groups . these observations , along with the consideration that only a subset of patients with diabetes develops dn , drove the search for the genetic determinants of dn susceptibility . one of the most consistent annotations in the field is probably the genetic variation on chromosome 18 . in 2002 , a family - based linkage analysis performed in t2dn turkish families and affected sibling pairs of pima indians reported a strong evidence for the localization of a dn susceptibility locus mapping to chromosome 18q22.3 - 23 . researchers were not able to pinpoint the precise susceptibility gene but the same locus was also detected in a t2dn african american population . later studies on chromosome 18 led to the identification of a susceptibility marker within the carnosine dipeptidase 1 ( cndp1 ) gene , and it was also described how the shortest allelic form of the cndp1 gene was more common in the absence of nephropathy . the cndp1 gene encodes the secreted enzyme serum carnosinase that degrades carnosine , a protein controlling the formation of age molecules . as previously discussed similar results were obtained in a meta - analysis study when investigating a multiethnic population with t2d - esrd ; a recently published meta - analysis confirmed the association of the carnosinase d18s880 microsatellite polymorphism with dn susceptibility in a t2d caucasian population although no significant association with t1dn could be found . in a very recent candidate - gene driven study , palmer et al . performed a genotyping of several snps across 22 dn candidate genes in a large cohort of african americans with t2d and esrd . after adjustment for the apol1 g1/g2 alleles , known to be associated with nondiabetic esrd in this population , the most significant signals were observed downstream of the cndp1 gene , at chimerin 2 ( chn2 ) locus and within angiotensin ii receptor type 1 ( agtr1 ) gene . in another work , to investigate the impact of oxidative stress on disease initiation , the polymorphic variants of 7 genes involved in the antioxidant defense were evaluated : sod2 , p22 phox , cat , mpo , gstp1 , gstt1 , and gstm1 . despite the commonly recognized link between oxidative stress and diabetes , authors claim that no association could be found in caucasian t2d patients . in one of the first dn genome - wide genotyping studies , authors reported the engulfment and cell motility 1 ( elmo1 ) gene on chromosome 7p as a likely candidate for disease susceptibility in a japanese patients cohort with t2d . in a cellular system engineered to overexpress elmo1 , they furthermore observed increased expression of extracellular matrix ( ecm ) protein genes and decreased expression of matrix metalloproteinases . finally , recent data from a meta - analysis study suggests the elmo1 association with dn exclusively in the t2d asian subgroup . in a population of pima indians with t2d , the gwas of over 100,000 snps led to the identification of several loci with significant association for esrd susceptibility , with the strongest signal located in the intronic region of the of pvt1 gene . some of these findings were also replicated in an ethnically different population with t1d . in a gwas performed on a large cohort of african americans with t2d and esrd , five gene regions with evidence of association with dn were detected , nominally , sash1 , rps12 , auh , msrb3-hmga2 , and limk2-sfi1 . some of these snps however were later found to contribute to all - cause esrd . in order to establish a comprehensive , well - defined dna biobank for the genotyping of dn in t1d in particular , the first results of this genome - wide scan were reported by pezzolesi et al . in 2009 . authors claimed that although no snp achieved genome - wide significance , strong association was found near the 4.1 protein ezrin , radixin , and moesin [ ferm ] domain containing 3 ( frmd3 ) locus and near the cysteinyl - trna synthetase ( cars ) locus . further studies confirmed the 9q21.32 region ( upstream of frmd3 ) as a susceptibility locus for t2dn in several unrelated study populations [ 20 , 21 ] . despite all the effort currently invested into this field of research , at present it is still impossible to predict those diabetic patients with a higher risk for developing dn . indeed , in almost all the studies published so far on dn susceptibility , diagnosis was based almost exclusively on the presence of hyperglycemia and proteinuria ; therefore , it is not possible to exclude that the inconsistencies among the findings could be linked to a misclassification of the renal damage in the diabetic population . the transcriptome represents the part of genome that is transcribed and includes both coding and noncoding rna molecules . when studying the transcriptome , as for genetic studies , either targeted or genome - wide approaches can be used . rna - sequencing ( rna - seq ) , arrays , and quantitative pcr ( qpcr ) are the techniques employed routinely to assess rna expression . qpcr is very sensitive and even subtle changes can be detected precisely ; arrays on the other hand are very high - throughput but also less sensitive . rna - seq takes advantage of the recent next - generation sequencing platforms and it has rapidly become the method of choice for transcriptome profiling . the main advantages of rna - seq are its very high resolution ( down to a single nucleotide ) , its potential to detect novel transcripts , its ability to measure either primary transcripts or spliced mature mrnas . given the plethora of gene expression data available in the literature , only the research on dn kidney tissue or urine will be discussed . all the coding and noncoding rna markers cited in this paper the first transcriptomic signature of dn kidney was published in 2004 . using an array - based approach , baelde et al . the results of this genome - wide analysis indicated that 96 genes were upregulated in t2dn , including aquaporin 1 ( aqp1 ) , calpain 3 ( capn3 ) , hyaluronoglucosidase , and platelet / endothelial cell adhesion molecule ( pecam-1 ) . over 500 genes were downregulated , including bone morphogenetic protein 2 ( bmp2 ) , vascular endothelial growth factor ( vegf ) , fibroblast growth factor 1 ( igf-1 ) , insulin - like growth factor binding protein 2 ( igfbp-2 ) , and nephrin . in the same manuscript , authors confirmed reduced expression of vegf and nephrin in renal biopsy specimens from additional dn patients at both the protein and rna levels . to explain the existing inconsistencies between human and murine progressive dn , microdissected biopsies from controls , early and progressive t2dn patients underwent global gene expression profiling through microarray hybridization . preliminary results , later confirmed using qpcr , revealed an upregulation of jak-2 and a compromised expression of several members within the jak / stat signaling pathway which could not be detected in either db / db c57blks or diabetic stz - treated dba/2j mice . more recently , woroniecka et al . performed the transcriptome analysis on microdissected kidney biopsies from dn patients , healthy living transplant donors , and patients undergoing tumor nephrectomies ( analyzing the histologically normal kidney tissue ) . the microarray - derived expression profiles indicated that several podocyte - specific transcripts were downregulated , including plce1 , ptgds , nphs1 , nphs2 , synpo , pla2r1 , wt1 , clic5 , and podxl . glomerular transcripts showing upregulation included igh , c3 , col1a2 , cxcl6 , and col6a3 . in the tubular compartment instead , authors detected increased expression of different transcripts including igh , igl , col1a2 , and col3a1 . several reports analyzed the gene expression of both the glomerular and tubular compartments of t2dn kidney biopsies . among the mrna transcripts detected as enriched in the glomerular compartment of t2dn individuals are mrp8 , wnt1 , wnt2b , wnt4 , wnt6 , wnt16 , dkk3 , and lef1 , pkc , fsp1 , angptl2 , and ace . decreased expression for ace2 , vegf [ 64 , 106 ] , ctgf , nephrin , podocin , and wt1 tubule - rich renal biopsies from patients with t2dn , ihg-1 , il6 , ccl2 cd68 , and ccr5 were increased , while tlr4 was overexpressed in both glomeruli and tubules of microalbuminuric and overt dn . using biopsy material collected by the european renal cdna bank , the gene expression of tubulointerstitial mrna from human dn kidneys was compared to that of living donors , cadaveric donors , and patients with minimal change disease through a combined microarray profiling and qpcr validation approach . results indicated dysregulation of specific nf-b targets , highlighting the existence of an inflammatory signature characteristic of progressive dn . eight genes in particular were induced in t1dn and t2dn relative to controls : ccl5/rantes , cxcl10/ip10 , edn1 , vcam1 , hla - a , hla - b , ifnb1 , and b2 m . further work performed using the european renal cdna bank material highlighted additional mrna transcripts as dysregulated in t2dn kidney when compared to normal tissue . within the glomerular compartment in particular , nrp1 and nrp2 were significantly lower in t2dn , while smpdl3b was increased . within the tubulointerstitial compartment , upregulation of mmp7 and fgf-2 , of the unfolded protein response genes hspa5 , hyou1 , and xbp1 and of the apoptosis - related genes trail and opg , the expression of several transcripts was assessed on whole t2dn kidney tissue . upregulated mrnas included hdac2 , hdac4 , and hdac5 , b7 - 1 , stat1 , tnfaip8 and tipe2 , prkc - beta , vegf , uii and ut , pdgf - a and pdgf - b , lox1 , ldlr , and cd36 , jagged1/hes1 , and gremlin [ 45 , 46 ] . decreased transcription was detected for autophagy - related genes beclin 1 , lc3 [ 33 , 34 ] and atg7 , cxcl16 , abca1 , abcg1 , and apoe , timp3 , foxo1 and foxo3a , atg5 , and atg8 , ankrd56 and entpd8 , and nephrin . in other works the study design was developed to compare t2dn with other glomerulopathies . using a qpcr based approach , the tubulointerstitial compartment isolated from kidney biopsies of both dn patients , living donors , and minimal change disease patients was profiled specifically for the expression of 202 candidate genes involved in molecular pathways contributing to dn progression . results showed a decreased expression of vegf and egf , while collagens i and iv , fibronectin 1 , and vimentin as well as matrix metalloproteinases 2 , 7 , and 14 and tissue inhibitor of metalloproteinases 1 and 3 were increased . in another study , increased irs2 mrna was detected in dn patients compared to controls , while no significant changes irs2 expression were present in biopsies from patients with focal - segmental glomerulosclerosis or membranous nephropathy . low expression of robo2 mrna was present in dn compared to nephrosclerosis , focal - segmental glomerulosclerosis , membranous nephropathy , and control pretransplant biopsies . a strong specific induction of col8a1 and col8a2 mrnas expression was found in both glomerular and tubular compartments of biopsies from patients with t2dn versus control pretransplant biopsies , benign nephrosclerosis , and focal - segmental glomerulosclerosis . finally , increased ace expression was observed in t2dn biopsies compared to benign nephrosclerosis , minimal change nephrotic syndrome , and lupus nephritis . aiming to develop a diagnostic tool for early dn diagnosis , zheng et al . designed a pcr - array platform to detect expression changes in 88 genes simultaneously and employed it in a pilot study where the urinary sediment of dn patients was assayed . authors found that several mrnas were significantly increased in dn compared to healthy controls , in particular , notch3 , actn4 , cdh2 , ace , fat1 , col4a1 , synpo , and twist1 . increased mrna levels of podocalyxin , cd2-ap , nephrin , wt-1 , -actinin 4 podocin , and synaptopodin [ 28 , 29 ] were found in the dn group compared with controls . finally , in another work , authors claim that urinary expression of nephrin and podocin was useful for distinguishing diagnostic groups ( iga nephropathy , minimal change disease , and membranous nephropathy ) as well as predicting renal function decline . until a few years ago , the molecular profiling of dn was mainly focused on the characterization of mrna transcripts . over the last decade however , much interest has converged toward the profiling of noncoding rna ( ncrna ) molecules . the ability of ncrnas to modulate gene expression along with the discovery that they can be detected in biofluids and are fairly stable makes them ideal biomarker candidates . micrornas ( mirnas ) are probably the most studied ncrnas ; they are short , single - stranded , highly conserved , and tissue - specific . the partial match binding feature allows mirnas to bind hundreds of targets simultaneously ; accordingly the dysregulation of even one single mirna molecule can profoundly influence the gene expression profile of the surrounding environment . for a complete review on mirnas biogenesis and function refer to [ 107 , 108 ] . in the field of dn , the majority of mirna 's profiling studies was performed on cellular and animal models . more recently , with the surprising discovery that mirnas can be released and carried into the extracellular environment , different body fluids are being characterized in their mirna 's content . initially identified in a mice model of dn , mir-192 , along with mir-377 , mir-337 , and mir-129 , was later discovered as being enriched in human mesangial cells ( mcs ) exposed to high glucose . interestingly , when assessing mir-192 in human dn kidney , expression levels not only are reduced but also inversely correlate with severity of kidney disease , raising once again the issue about the appropriateness of the currently available animal models for dn . mir-21 has recently emerged as a marker for fibrosis in many complications [ 110 , 111 ] ; unsurprisingly , increased mir-21 expression was also detected in human t2dn kidney biopsies relative to healthy controls . except for the previously mentioned dn kidney profiling from krupa et al . , the array - based mirnome analysis of t2dn kidneys was recently published by huang et al . and uncovered mir-155 and mir-146a enrichment in these samples . these two are the only works describing the mirnome of human dn kidney ; noteworthy , the existence of strict renal biopsy policies in most nephrology clinics might be a limiting factor in terms of sample collection and availability . in parallel , the urgent need for novel biomarkers of diagnosis and progression shifted priority to the profiling of more accessible samples , such as biological fluids . using a qpcr based approach , argyropoulos et al . were the first to perform the urinary mirna profiling of t1d patients with and without proteinuria . results showed that mir-323b-5p , mir-221 - 3p , mir-524 - 5p , and mir-188 - 3p were underexpressed in albuminuric relative to nonalbuminuric patients , while mir-214 - 3p , mir-92b-5p , hsa - mir-765 , hsa - mir-429 , mir-373 - 5p , mir-1913 , and mir-638 were overexpressed . in a similar study performed on the rna content of urinary exosomes , authors showed that mir-130a and mir-145 were enriched in t1d patients with microalbuminuria compared to normoalbuminuric subjects , while mir-155 and mir-424 were reduced . in a work aimed to determine the urinary levels of all mir-29 family members ( mir-29a , mir-29b , and mir-29c ) , mir-29a was significantly increased in albuminuric t2dn patients compared to normoalbuminuric patients and it also correlated with the degree of albuminuria . in the work from szeto et al . , when comparing the urinary sediment of patients with either iga nephropathy , dn , or hypertensive nephrosclerosis , mir-15 was decreased in dn samples compared to other groups . similarly , in another work authors found that mir-192 levels were reduced in urinary sediment of dn patients compared to both healthy controls and patients with either minimal change nephropathy , focal glomerulosclerosis , membranous nephropathy , or other diagnosis groups . mirnas expression was also measured in venous blood from t2d han chinese patients with and without albuminuria . using a microarray - based approach , authors identified several differentially expressed mirnas in the different study population and confirmed mirna let-7a downregulation using qpcr . very interestingly , authors also observed how the distribution of a specific variant within let-7a ( rs1143770 ) was significantly higher in diabetic patients ( with and without albuminuria ) relative to control subjects . finally , dysregulation of a new class of noncoding rna molecules has emerged as being potentially involved in different complications , including kidney disease . among these noncoding rna molecules , recent effort aims to characterize the so - called long noncoding rnas ( lncrnas ) . this led to the initial assumption that lncrnas were not biologically relevant . today we know that lncrnas contain individual domains and structural motifs that allow them to specifically associate with dna , rna , and/or protein and thus regulate their function . as previously discussed , multiple experimental evidence , from different ethnic populations , suggested a link between diabetic kidney disease and genetic variants within the pvt1 locus [ 22 , 23 ] . pvt1 , whose increase is significant in mesangial cells stimulated with high glucose , can induce the expression of plasminogen activator inhibitor 1 ( pai-1 ) and transforming growth factor beta 1 ( tgf-1 ) . noteworthy , six different mirnas are encoded within the pvt1 gene ; therefore , authors investigated whether an alteration in pai-1 and tgf-1 gene expression was ascribable to the pvt1 lncrna transcript itself or whether it was the result of a mutation within the mirnas encoded in the pvt1 gene . results showed that both pvt1 lncrna and mir-1207 - 5p were induced by high glucose independently and they both contributed to ecm accumulation in the kidney . the term epigenetics refers to all those dynamic structural changes that , while not resulting from an alteration in the dna sequence , affect gene expression and can be inherited . epigenetic modifications , such as dna methylation , histone methylation , and histone acetylation , modify the accessibility of the chromatin and thus modulate transcription . they are responsible for the phenotypic differences within cell types and explain why the gene expression profile of an organism can change so profoundly during development . unlike genetics , epigenetics is highly susceptible to influences from the environment ; therefore , the understanding of its regulatory machinery offers an incredible opportunity for disease management . the study of epigenetics in diabetic kidney disease is still in its embryonic phase although increasing evidence indicates metabolic memory as a consequence of long - lasting epigenetic modifications contributing to dn progression . in 2007 geisel et al . analyzed the promoter methylation of the stress response protein p66shc , previously shown to increase susceptibility to oxidative stress and atherosclerosis . in peripheral blood mononuclear cells isolated from esrd patients and control subjects , authors demonstrated that increased p66shc expression in esrd group was linked to a significant reduction in the methylation of its promoter region . using an array based approach , the genome - wide promoter dna methylation of 192 t1d patients was analyzed searching for any possible association with dn . the analysis was conducted using dna extracted from peripheral blood cells as these include the t cell population responsible for islet beta cells destruction in t1d . importantly , among the several cpg islands showing correlation with dn development , results uncovered one in particular ( rs10081672 ) , located upstream of the unc13b gene . additionally , this region is in strong linkage disequilibrium with rs13293564 , a variant associated with dn susceptibility . importantly , depending on which allele is present in rs10081672 , a cpg site is either created or abrogated , thereby affecting transcription factor binding . in another work , the genome - wide dna methylation of diabetic patients with esrd and diabetic patients without nephropathy was compared with the aim to identify novel disease biomarkers for noninvasive diagnosis . patients ' saliva was employed as starting material for dna extraction while the study population included african americans and hispanic individuals . results highlighted differential methylation at two or more cpg sites in 187 genes between the two groups . interestingly , many of these genes are involved in inflammation , oxidative stress , ubiquitination , fibrosis , and drug metabolism , and some in particular are even known for their genetic association with dn , suggesting once again a very close connection between genetic dysregulation and epigenetic dysregulation in the pathogenesis of dn . a recent paper from hasegawa et al . demonstrated that sirt1 , a protein deacetylase that targets histones and transcription factors , is reduced in stz - treated mice . using a transgenic mouse model authors also elucidated the interaction between sirt1 expression and cpg methylation of cldn1 , a gene encoding for the protein claudin-1 . claudin-1 is a tight junction protein involved in cell - to - cell adhesion and authors suggest that its epigenetic - mediated induction is responsible for podocyte effacement and proteinuria . in support of this hypothesis authors also revealed the correlation between proteinuria and sirt1 expression in human dn kidney . finally , reddy et al . elegantly demonstrated the link between the protective effect of angiotensin ii receptor antagonist , losartan , and its ability to reverse specific epigenetic modifications in the glomeruli of diabetic db / db mice . all these experimental evidences show that epigenetics holds the potential to allow a temporary and reversible manipulation of the gene expression , conferring protection from disease progression . the proteome probably represents the most complete expression of the potentialities of a living organism since it focuses on the set of proteins , expressed by the genome , that regulate biological and metabolic cell function . the proteomics , formally defined as the massive and mass spectrometric - based analysis of the proteome , is a complex and interdisciplinary matter requiring expertise spanning from chemistry to biology and bioinformatics , in order to reveal the meaning of complex protein datasets of a biological sample in physiological and pathological conditions . unlike genomics studies , based on the analysis of biological samples that may be expanded artificially making complex studies from little starting material possible , proteomics requires a larger amount of starting sample that can be easily available in biological fluids rather than in the tissues or cells . for this reason , proteomic studies in nephrology are more oriented to the analysis of biological fluids and have led , in the last decade , to the identification of a number of putative biomarkers that are expected to enter shortly into the clinical practice . in the next paragraphs we will discuss the main application of proteomics to the identification of new potential biomarkers of dn in kidney tissues and biological fluids with a special emphasis on the new emerging potentialities of the post - translational modifications ( ptms ) screenings . glomerular damage plays a critical role in the onset of dn making this renal compartment a key target for proteomic investigation . however , only few proteomic studies have been carried out on isolated glomeruli since , in general , renal biopsy is rarely carried out on diabetics patients and the number of isolated glomeruli , when starting form biopsy material , is too scarce to produce homogeneous preparations of individual specimens and to extract adequate glomerular protein amounts for deep proteomic studies . recent methodological improvements have now permitted the extraction of intact and unmodified proteins from formalin fixed paraffin embedded ( ffpe ) samples thus making available the use of vast archive of kidney tissues for proteomic analysis . proteomic analysis of isolated glomeruli , obtained by laser capture microdissection ( lcm ) , allowed the identification of over 100 differentially expressed tissue proteins between dn and nondiabetic glomeruli . notably , the results of this study probably underestimates the differences of the glomerular proteome since it was carried out on ffpe tissues derived from autopsy cases undergoing postmortem proteolysis . however , among differently expressed proteins , nephronectin , a protein implicated in the assembly of extracellular matrix and nephrogenesis , was confirmed as differently expressed in dn tissue specimens using immunohistochemistry . a similar study reported increased expression of c3 and the membrane attack complex ( c5b-9 ) and a marked reduction of podocyte - associated proteins and antioxidant proteins in dn . even if these proof of concept studies demonstrate the usefulness of ffpe tissue proteomics , the potentialities of this approach are still prevented by the poor availability of tissue specimens that limits the identification of the key molecular events involved in the onset and progression of dn . biofluids encompass any liquid originating from inside the bodies of living organism . among the body fluids proteomics colleagues reported , in urine of t1d patients with dn , a panel of 65 urine biomarkers , mainly composed of collagen fragments , that was further validated in a multicentre independent cohort of t2 dm patients [ 124 , 125 ] . expanded the 65 peptides classifier to 273 and demonstrated its ability to predict the occurrence of the microalbuminuria in t1d and t2 dm normoalbuminuric patients [ 126 , 127 ] . these data were recently confirmed in another independent study that specifically identified subsets of urine biomarkers able to predict to the transition from normo- to microalbuminuria or from micro- to macroalbuminuria indicating that the appearance of collagen fragments in urine of t2 dm patients may have both diagnostic and prognostic values . lc / ms / ms analysis of 22 t1d normoalbuminuric patients developing microalbuminuria after 6 years median follow - up allowed identifying a set of potential predictive biomarkers that were further validated by elisa assay . of note , the introduction of these proteomic biomarkers ( thp , progranulin , alpha-1-glycoprotein , and clusterin ) into the baseline model that included diabetes duration , baseline albumin excretion rate ( aer ) , hba1c , cystatin c , and uric acid improved the prediction of renal function worsening from 84% to 89% . jin et al . used isobaric tags for relative and absolute quantification ( itraq ) and lc / ms / ms to quantify and identify a set of urinary proteins differentially excreted between normoalbuminuric and microalbuminuric t2 dm patients . three protein biomarkers , namely , alpha-1-antitrypsin , alpha-1-acid glycoprotein 1 , and prostate stem cell antigen , were included in a multiplex assay that was able to correctly classify normoalbuminuric and microalbuminuric t2 dm patients with about 92% accuracy . two mass peaks corresponding to b2-microglobulin and ubiquitin ribosomal fusion protein that were selectively and differently excreted in nephropathic diabetic patients . we further refined this study by selecting only diabetic patients with biopsy - proven kimmelstiel - wilson lesions and identifying both urinary b2-microglobulin and free ubiquitin as specific biomarkers of diabetic glomerulosclerosis over other nondiabetic kidney lesions . although the overall analysis of the urine proteome is up to now the most used way to search for disease - specific biomarkers , the future of this matter will be the analysis of well - purified proteins subfractions since it may provide more detailed information about simplified proteomes and potentially improve the knowledge of specific pathways . until few years ago , the most useful way to reduce the proteome complexity was the selective antibody - based depletion of the most abundant proteins . in the last few years , the enrichment of post - translationally modified proteins has begun a new strategy to highlight functionally interesting proteins . protein phosphorylation is a key player in the regulation of most cell pathways ; thus , phosphoproteome screening of urine samples may represent a precious source of information about deregulated cell processes in many kidney diseases including dn . however , up to now , urine phosphoproteome analysis has not been applied yet to soluble proteins in dn and other ckd probably because most of the historical collections of urine samples have not been prepared and stored in presence of phosphatases inhibitors that , preventing the liability of this ptms , may ensure more reproducible results . on the contrary , the analysis of the microvesicular fraction ( i.e. , exosomes ) that originates from renal epithelial cells and are released into urine may be , at the moment , more useful to study this kind of ptm as the presence of the exosomes ' membrane may preserve ptms by protecting their protein content from spontaneous degradation and dephosphorylation by proteases or phosphatases , respectively . have already published the first proteomic study on urine exosomes of dn patients demonstrating the potentiality of this microvesicular screening for identifying dn specific biomarkers . specifically , 3 over the 25 most significant differently expressed proteins , namely , voltage - dependent anion - selective channel protein 1 ( vdac1 ) , isoform 1 of histone - lysine n - methyltransferase mll3 , and alpha-1-microglobulin / bikunin precursor ( amb ) , were also validated . of note , mll3 , a specific tag for epigenetic transcriptional activation , was detected only in dn exosomes , thus emphasizing the potential importance of epigenetic mechanisms in the pathophysiology of dn . it is reasonable to think about the forthcoming application of the exosomes ' phosphoproteomics as a new way to identify specific deregulated patterns in kidney diseases . as for phosphoproteomics also , only one paper has applied this approach to the study of ckd identifying a number of urinary proteins involved in immune / stress response and many biological functions like homeostasis , platelet degranulation and coagulation , transport , and secretion . due to the importance of the glycoproteomics in cell - cell interaction and signalling cascades , it is reasonable that many further studies will be planned in the next year to understand , by screening this specific subset of proteins , the molecular mechanisms involved in damage progression of specific nephropathies including dn . interestingly , the usefulness of the glycoproteomics for the diagnosis of dn has been recently reported in plasma where thirteen significantly upregulated glycoproteins were described in dn patients compared to t2 dm patients without nephropathy . among these , increased plasma levels of glycated lumican , vasorin , and retinol binding protein-4 were validated by immunoblotting and showed potential specificity for dn . by using a different proteomic strategy , kim and coworkers reported that increased plasma levels of glycated pedf , apolipoprotein j precursor , hemopexin , immunoglobulin mu heavy chain , and immunoglobulin kappa chain correlated with poor glycaemic control in t2 dm patients while glycated prekallikrein and complement factor c4b3 correlated with microalbuminuria and other glycated proteins such as hemopexin precursor , serine proteinase inhibitor , alpha-1-antitrypsin , and haptoglobin - related protein were associated with dn . these studies confirmed the potentiality of the plasma glycoproteome for the identification of reliable biomarkers of dn and their importance is emphasized by the consideration that the overall analysis of serum / plasma proteome is challenging because the candidate biomarkers are generally present in trace amounts . of note , there is an alternative way to reduce the complexity of this biological fluid , namely , the prefractionation of the samples , achieved by several known strategies before the analysis , that allow removing the large background of nonrelevant and abundant proteins and may favour the discovery of potential candidate biomarkers . up to now only few studies have used this approach to analyse the serum [ 89 , 132 ] or plasma proteome of t2 dm patients . these studies have reported extracellular glutathione peroxidase ( egpx ) and apolipoprotein ( apoe ) as potential diagnostic biomarkers of dn and vitamin d - binding protein ( dbp ) as early biomarker of renal damage in t2 dm . overall many independent studies are showing an increasing number of new biomarkers that are potentially useful for both early diagnosis and monitoring of the disease and to understand ever more deeply its pathogenesis . metabolomics is a systematic evaluation of small molecules ( i.e. , metabolites ) that may provide fundamental biochemical insights into disease pathways , drug toxicity , and gene function . metabolomics profiling is generally carried out by nuclear magnetic resonance ( nmr ) and ms - based profiling each with advantage and limitations . two main strategies may be adopted for metabolomics analysis of biological samples : targeted and untargeted profiling . the targeted profiling focuses only on sets of few metabolites generally included in specific metabolic pathways while untargeted analysis provides a comprehensive evaluation of the metabolome without any a priori hypothesis on the metabolic pathways . targeted analysis is an essential tool for the investigation of biological mechanisms rather than for biomarkers discovery ; in fact it is a quantitative approach that allows quantification of each metabolite of an interested metabolic pathway through the use of isotope - labelled standards . untargeted approach is instead more suitable for biomarker discovery since the whole metabolic profile of cases and controls may allow identification of disease - correlated biomarkers . as obvious , the latter approach needs , as for proteomics , further data analysis through supervised statistical methods in order to construct disease - specific metabolomics classifier further sequenced by mass spectrometry . in the last years , the optimization of the separation techniques has allowed the selectively purification of specific class of metabolites such as phospholipids and fatty acids , leading to the development of new more focused untargeted analysis such as phospholipidomics . as for proteomics , most of the metabolomics studies have been carried out on biofluids , namely , urine and serum / plasma . urine metabolomics may offer direct insights into biochemical pathways linked to kidney dysfunction since a variety of metabolites are concentrated by the kidney and excreted in urine . used targeted analysis to investigate the urinary excretion of 94 metabolites in healthy subjects ( hs ) and t2 dm patients with ( dm+ckd ) or without ( dm - ckd ) ckd . thirteen metabolites differently excreted between t2 dm patients and hs were also useful to differentiate dm+ckd from dm - ckd . interestingly , 5 out 13 metabolites were differently excreted between dn and other ckd , thus being specifically associated with the diabetic kidney disease while 8/13 reflected metabolic changes shared by diabetic and nondiabetic ckd . most of the less excreted metabolites in dn group were water soluble organic anions and functional analysis correlated them with impaired mitochondrial function in dn . very recently , pena and colleagues carried out an untargeted analysis of urine and plasma metabolome by gc - ms and reported the possible usefulness of a set of metabolites to predict the development of dn on top of the traditional renal risk markers , namely , baseline urinary albumin excretion and baseline estimated glomerular filtration rate . in this prospective study , 24 normo- to microalbuminuria case / controls pairs and 21 micro- to macroalbuminuria case / controls pairs were enrolled . the metabolomic profiles of micro- to macroalbuminuria case / control pairs show significant differences while normo- to microalbuminuria pairs remained unchanged . specifically they reported two plasma metabolites ( butenoylcarnitine and histidine ) and three urine metabolites ( hexose , glutamine , and tyrosine ) significantly differentially excreted in microalbuminuric patients prone to develop macroalbuminuria . the area under receiving operating characteristic ( roc ) curve arising from the integration of these urine and plasma metabolites to a reference model based on baseline egfr and urine albumin excretion passed form 84% to 99% correct prediction . although these results appear impressive , as the authors suggest , they still need to be managed with care until a validation study on larger and independent cohorts will be set up . some of the identified metabolites may have direct link with the pathopysiology of diabetes and its chronic complications since , for example , butenoylcarnitine plasma accumulation has been related to the excessive yet incomplete mitochondrial oxidation of fatty acids , possibly attributable to a lower mitochondrial number and reduced oxidation capacity in t2d tissues while histidine , a modulator of inflammation and oxidative stress , may be correlated with impaired inflammation and oxidative stress in t2 dm and ckd patients . it is worth noting that both studies stressed the importance of mitochondria dysregulation in the pathogenesis of dn . urine metabolomics has been also applied to type 1 diabetic patients in order to identify predictive biomarkers of renal function worsening . metabolite profile of baseline 24 h urine samples of 52 type 1 diabetic patients ( 26 stable normoalbuminuric and 26 progressed toward microalbuminuria in 5.5 years ' follow - up ) was carried out by lc / ms and gc - ms . multivariate logistic regression analysis of gc - ms and lc / ms dataset showed 65% and 75% predictive power after cross - validation , respectively . twenty - one and 14 compounds showed a significant contribution to the logistic regression model based on gc - ms and lc / ms dataset , respectively . most of the identified gc - ms compounds were carboxylic compounds , acidic metabolites , and endogenous amino acids not showing a documented direct relation to dn while lc - ms dataset reveals specific compounds related to impaired fatty acids metabolism , detoxification system , and gut microbiome . serum and plasma metabolomics has been carried out of both whole samples and specific subfractions . marrachelli and coworkers performed both genomic and metabolomic screening of over 1500 caucasian t2 dm patients , characterized the serum metabolome profile of the microalbuminuric patients by nuclear magnetic resonance ( nmr ) , and correlated it with specific genotypes , thus reporting a potential predictive value of the genotype on the onset of microalbuminuria in t2 dm . furthermore , hirayama et al . reported , in t2 dm patients , 19 serum metabolites including creatinine , aspartic acid , -butyrobetaine , citrulline , symmetric dimethylarginine ( sdma ) , kynurenine , azelaic acid , and galactaric acid that were positively correlated with albuminuria and negatively with egfr . multiple logistic regression , carried out on identified metabolites , recognized 4 features , namely , aspartic acid , azelaic acid , galactaric acid , and symmetric dimethylarginine ( sdma ) as relevant for the model and allowed correct identification of dn patients with about 75% accuracy . zhang et al . carried out serum metabolomic profiling of 8 dn patients , 33 type 2 diabetes mellitus ( t2 dm ) patients , and 25 healthy volunteers in order to investigate the presence of dn biomarkers . importantly , they reported significant changes of leucine , dihydrosphingosine and phytoshpingosinewere specifically in the dn cohort , thus suggesting the perturbations of amino acid metabolism and phospholipid metabolism as key events in diabetic disease . other authors have instead investigated specific subfractions of the metabolome , namely , compounds linked to purine and pyrimidine metabolism , phospholipids , and fatty acids . xia et al . standardized an analytical method for analysis and quantification of purine and pyrimidine metabolites in dn patients and matched healthy controls . according to the well - established association of the purine and pyrimidine metabolic pathway with the development of the dn , they could assess that uric acid , xanthine , and adenosine were significantly increased in dn patients ( especially in those at stage v according to mogensen classification ) while inosine is reduced probably as a result of the adenosine deaminase inhibition that catalyzes inosine formation from adenosine . several phospholipids ( pls ) , significantly upregulated or downregulated in disease models , have been already recognized as potential biomarkers of t2 dm or dn [ 138 , 139 ] . comprehensive and quantitative analysis of plasma pls , such as phosphatidylethanolamine , phosphatidylglycerol , phosphatidylcholine , phosphatidylinositol , phosphatidylserine , sphingomyelin , and lysophosphatidylcholine , may selectively distinguish t2 dm from dn patients . targeted quantification of the phospholipids revealed proportional decrease of phosphatidylinositol and linear increase of sphingomyelin in dn patients . although the molecular pathogenetic mechanisms leading to impaired metabolism of phospholipids are not clear , the authors suggest that reduced phosphatidylinositol may reflect increased sorbitol pathway activation in t2 dm while increased sphingomyelin may depend on glucocorticoids - mediated sphingolipids metabolism . also plasma fatty acids ( fas ) may have a direct impact on the occurrence and development of diabetes since their abnormal accumulation in parenchymal cells of multiple tissues , called lipotoxicity , has been suggested as a trigger of t2 dm and its chronic complications . specific metabolomics screening of fas , namely , lipidomics , may contribute to the understanding of this disease . han and colleagues reported a standardized method based on gas chromatography - mass spectrometry ( cg - ms ) useful for the specific assessment of nonesterified and esterified fatty acids ( nefas and efas , resp . ) . lipidomics screening of 150 patients including diabetics with and without nephropathy showed high discrimination power on different stage of dn . disease progression was specifically correlated with plasma levels of arachidonic acid that is involved in the anabolism of prostaglandins , thus suggesting a key role of the inflammatory processes in the progression of dn . as genetic studies conducted so far are still inconclusive , it is difficult to envisage a common genetic basis for the development of dn . quite possibly a number of environmental factors contribute significantly toward the evolution of the diabetic patient to this specific complication . however , there is no doubt that , from the earliest stages of the disease , many molecular changes , observed at the transcriptomics , proteomics , and metabolomics level , anticipate the onset of a clinical phenotype and may allow us to reconstruct in detail the pathogenetic basis of kidney damage in t2 dm . although new omics challenges such as the analysis of the protein post - translational modifications and of multiprotein complexes , mimicking what naturally happen in intracellular behavior , will further broaden our understanding of the dn pathogenesis , we are already able to identify the common thread that unites all the disparate molecular changes described in the literature by performing bioinformatic - based analysis of genes , transcripts , proteins , and metabolites described so far . we can envisage that the selection of specific omic biomarkers and clinical phenotypes might lead to a better stratification of patient 's specific type of renal damage in t2 dm and might allow the identification of patients that progress or respond to a specific therapy . to accomplish this task and go forward , however , there is an urgent need to build up disease - specific platforms containing personal , clinical , and omics profiles that will allow the full potential application of systems biology analysis and the development of specific disease phenotype models . we can expect in the next future the development of new paradigms of renal damage in t2 dm that will contribute to defining of the road to the molecular medicine as a global , organized approach applicable to dn as well as to other relevant renal conditions .

background : chlorhexidine ( chx ) is considered as a gold standard of antimicrobial rinses . various herbal oral rinses are available in the market . however , little is known of its effectiveness.aim:the aim of this study was to evaluate the clinical changes after the usage of herbal oral rinse and 0.12% chx.materials and methods : in a randomized clinical trial , 76 patients with dental plaque - induced gingivitis were assigned to group i ( herbal oral rinse - hiora ) and 76 patients with dental plaque - induced gingivitis to group ii ( 0.12% chlorhexidine - peridex ) . gingival index and plaque index scores were recorded at baseline and 21 days after scaling.results:intragroup comparison in both groups showed that plaque index and gingival index scores were statistically significant after 21 days as compared to baseline . intergroup comparison showed that plaque index scores and gingival index scores were statistically significant in group ii as compared to group i.conclusion:when herbal oral rinse was compared to 0.12% chx , 0.12% chx mouth rinse effectively reduced the clinical symptoms of plaque - induced gingivitis . diabetic nephropathy ( dn ) , a microvascular complication occurring in approximately 2040% of patients with type 2 diabetes mellitus ( t2 dm ) , is characterized by the progressive impairment of glomerular filtration and the development of kimmelstiel - wilson lesions leading to end - stage renal failure ( esrd ) . the causes and molecular mechanisms mediating the onset of t2 dm chronic complications are yet sketchy and it is not clear why disease progression occurs only in some patients . we performed a systematic analysis of the most relevant studies investigating genetic susceptibility and specific transcriptomic , epigenetic , proteomic , and metabolomic patterns in order to summarize the most significant traits associated with the disease onset and progression . the picture that emerges is complex and fascinating as it includes the regulation / dysregulation of numerous biological processes , converging toward the activation of inflammatory processes , oxidative stress , remodeling of cellular function and morphology , and disturbance of metabolic pathways . the growing interest in the characterization of protein post - translational modifications and the importance of handling large datasets using a systems biology approach are also discussed .

in terms of phylogeny , aggression is among the oldest of evolved behavior patterns ( blanchard and blanchard , in press ) . exemplars of aggression have been reported in animals without a central nervous system ; in a host of invertebrates ; and in each of the seven classes of vertebrates , including the most primitive ; agnatha , hagfish and lampreys ( malmqvist , 1983 ) . there is an emerging consensus that one major function , i.e. adaptive consequence , of aggression , across animal species , is resource control ( e.g. wilson , 1971 ) , with the further provision that aggression typically occurs in the context of competition from conspecifics over such resources . term enhancement of access to resources that are important for that species ( blanchard and blanchard , 1984 ; moynihan , 1998 ) . resources and their distribution are also major factors in the development of species - typical social systems ( rubenstein , 2009 ) , of which within - species aggressive behaviors are one , important , component . some species such as mice are particularly opportunistic and may show rapid and dramatic changes in social structure in accord with habitat alterations ( bronson , 1979 ; gray and hurst , 1997 ) , a flexibility that may stem in part from their long - term status as human commensals , with its strong requirement of rapid adjustment to host - initiated changes ( blanchard et al . in press ) . however , the ecological conditions under which most mammalian species have evolved are less variable than those associated with human habitations and human geographic movements , and the social systems evolving in most habitats appear also to be more conservative and resistant to change than are those of mice . evaluation of the rate of change in evolved characteristics of animals in response to important alterations in ecological conditions constitutes a difficult field of study , but some estimates may be obtained when a fossil record is available that permits information on relatively specific ecological changes . for example , it has been estimated that reductions in selective behavioral responsivity to rattlesnakes and in resistance to the venom of these snakes by california ground squirrels both decline over periods measured in the tens of thousands of years after rattlesnakes have disappeared from the squirrels habitats ( coss et al . , 1993 ) . as this example suggests , when ecological changes make some of the existing characteristics of species less adaptive than previously , the replacement of these by more adaptive characteristics can be a lengthy and variable process ; moreover , maladaptive as well as adaptive aspects of the emerging behaviors may be relevant to the patterns that ultimately emerge . with complex behavior patterns such as those involved in social relationships an even longer time - frame might be needed than for venom resistance and other purely physiological adaptations . however , following long - term exposure to diverse situations , even closely related species would be expected to show behavioral differences based on what is most adaptive in their particular habitats : rubenstein ( 2009 , p. 243 ) gives the example of plains zebras ( equus burchelli ) , living in an environment in which close proximity of food and water allow female zebras to consistently form groups , in turn permitting males to vie for harems in this home range . in contrast , grevy 's zebra ( equus grevyi ) inhabit locales where food and water are widely dispersed and scarcer , such that females forage alone and must travel between feeding and watering areas . in this species males form territories along the traveling routes , gaining breeding access to females that pass through or linger within the territory . in both the harem and territorial social systems , zebra males attempt to control access to females in breeding condition , but the time - frame and the location of these efforts vary with the conditions under which these animals have evolved . some of these considerations may be relevant to a question that was raised at the very beginning of the scientific study of the evolution of behavior ( darwin , 1871 ) : what is the basis for the evolution of the lion 's mane ? is this related to the social systems of lions , which are unique among felids ? there are about 40 species of felids , all stemming from a split from other stem - line carnivores about 1015 million years ago ( mya ) . animals that can be identified with existing species emerged over a period from about 1012 mya ( for ocelots ) to very recently , possibly within historic times . felids range in size from the black - footed cat ( about 3 lbs ) to the siberian tiger , weighing about 200 more , and are world - wide in distribution , excepting only antarctica and most remote islands . felid social systems are relatively similar across species : adult animals tend to be solitary except for females and their young , and amicable adult encounters tend to be connected with reproduction . a great deal is known of the specifics of aggression in felids , due in large part to the work of paul leyhausen , who worked with domesticated cats and with other felid species in captivity , at the max planck institute for behavioral physiology , at wuppertal . large felids , of the genus panthera have also been the subjects of extensive field work , which has generally affirmed leyhausen 's conclusions about conservation of many aggressive behaviors and facial expressions across felid species . briefly , leyhausen ( 1978 ) described intraspecific aggression in these animals as a behavior that is capable of producing great damage , as all felids have weapon systems that have evolved to facilitate their roles as predators but are used also in within - species fighting . leyhausen also indicated that aggression in felids is more regulated by effective defenses than responsive to the submission signals that are quite effective in reducing intra - group fighting in many canid species , such as wolves and dogs . by selecting animals and arranging situations , leyhausen was able to polarize attack and defensive behaviors in domesticated cat subjects , or , alternatively , to maximize attack motivations for both combatants . in a highly polarized attack - defend situation , the attacker stands tall and advances directly toward the victim while emitting low growls . the defending animal crouches down , or , with a mixture of aggressive and fearful motives , may assume the classic halloween cat stance with arched back , and erected hairs . as the attacker approaches contact , the defending animal may assume a contorted posture , half on its back but facing the other cat , enabling both fore- and hindpaws to be drawn up and opposed to the oncoming attacker . both fore- and hindpaws may lash out at the attacker and disembowelment of the attacker is possible , particularly if it attempts to reach over to bite at the partly supine defender 's nape . however , when both animals are highly motivated to attack , a frontal approach is typically utilized by both , resulting in animals facing each other and delivering forepaw blows largely toward the head and neck of the other . cat social systems provide a strong enabling factor in the dangerousness of aggression in these animals . because of their solitary life - style , there is little reason for a felid under attack from a member of its own species to stay and receive bites and blows from the highly developed weapon systems that are characteristic of all felid species . the only context in which fighting may be strongly adaptive is mating , where flight , even if successful in avoiding injury , results in a substantial reduction in a male 's extended reproductive fitness . given that felids , particularly the larger ones such as tigers and leopards , kill large prey , it might be assumed that fighting over such prey would constitute a frequent occasion for fighting . however , the solitary life - style of these animals also acts to reduce conspecific encounters over prey . lions are a different story . first , with the exception of the domesticated cat , which is not yet a truly separate species as it still freely interbreeds to produce fertile offspring with its wild ancestors ( driscoll et al . , 2007 ) , the lion ( panthera leo ) is the most recent cat species to emerge . while the earliest lion - like cat may date to the late pliocene , cave paintings indicate that males had no manes ( barnett et al . , 2006 ) . modern maned lions appear to have descended from a single population that arose about 200 thousand years ago , possibly in africa , and spread throughout the northern hemisphere , replacing earlier lion - like cats in europe as recently as 1015,000 years ago ( yamaguchi et al . , 2004 ) . although yamaguchi et al . ( 2004 ) suggest , based on a comparative analysis of food abundance factors in living species , that the earlier holartic cave lion might have been group - living as are modern lions , there is no direct evidence that this was the case . thus this particular mode of sociality , highly unusual for felids , may have been characteristic of holarctic lions ( panthera leo spelaea ) or , it may have evolved , along with the male lion 's mane , only in modern lions . the classic hypothesis for a relationship between group - living and the development of manes is that manes reduce the dangers associated with fighting within lion groups ( darwin , 1871 ; ewer , 1968 ; schaller , 1972 ; blanchard and blanchard , 1984 ) . insofar as group - living is adaptive , serious injuries resulting from fighting within the group would be maladaptive for both the attacker and the injured animal , encouraging the latter to leave the group or reducing its ability to join in protecting the group from attack by outsiders . this is a particularly important consideration for lions , in that take - over of prides by nomadic lions entails infanticide by the new pride males ( loveridge et al . , 2006 ) providing an extremely direct and strong adaptive consequence for success or failure at take - over , which in turn reflects the strength and numbers of the male cohorts on each side of the battle . a recent proliferation of nature documentaries plus the development of youtube and other online sources has made videos of fighting in a variety of animals available . there are literally dozens of lion fighting videos online , and some of these provide clear footage of adult males fighting , with continuity , in nonenclosed areas that appear to be the animals natural habitats . although the films were certainly selected for dramatic impact , and it is sometimes difficult to ascertain whether any external provocation might have been involved , it seems highly unlikely that the animals actual fighting behaviors were trained or shaped . moreover , a variety of animals and videotaping sources appear to have been involved , substantially reducing the possibility that the clips present a systematically biased view of the actual behaviors involved in serious fights in lions . the form of both attack and defense in male lions appears to be highly consistent across these clips , particularly demonstrating the mutual frontal attack seen in other felids . the only time that an attacked male voluntarily exposes its back to an attacker is when it is fleeing . even then , as the pursuer comes close , the pursued animal typically turns toward it , presenting its own weapons . even a young cub , attacked and ultimately killed by an adult male , turns to face its attacker , flipping on its back in a move reminiscent of defensive domestic cats , as contact becomes imminent . this view of male lions manes as protective has recently been challenged by findings regarding wound locations on wild males , females , and young ; by studies measuring approaches to dummy male lions with varying mane characteristics ; and by observations of maneless lions in the tsavo area of kenya . with reference to the first of these , west and packer ( 2002 ) examined sites of lion - inflicted wounds on a large sample of lions , sorting them into four regions ; neck / shoulder , as delineated by the perimeter of the mane ; face ; trunk ; and legs . they found that wounds were fairly evenly distributed over all four regions in females and subadults , and over three , excluding the area covered by the mane , in adult males . moreover , females and subadults were equally likely to survive wounds to each body part . these figures are not easy to reconcile with those of schaller ( 1972 ) who describes three direct observations of lions killing lions ( p. 189 ) : of these , two involved bites to the nape of the neck , while the third was a bite to the lower back , severing the vertebral column . a fourth example , not witnessed but examined while the victim was still alive , albeit expiring , involved a pride male with multiple deep wounds , including a broken saggital crest , and a penetrating wound to the chest , with tatters of his mane scattered over a 3 10 meter area ( p. 48 ) . these examples do not indicate that other sites are not involved in attacks of one lion on another , but they do suggest that bites to the head and nape , if they reach these targets , are likely to be especially lethal . moreover , in 259 observations of aggressive acts between female lions and cubs at kills , about 75% involved slaps at the head and neck , suggesting that these are indeed targets , albeit of blows that appear less likely to leave wounds ( schaller , 1972 , p. 134 ) . it might also be noted that wound sites do not necessarily have the same meaning as do targets : if , as in domestic cats , female and subadult lions have behavioral defenses that serve to protect the neck and shoulder by interposing teeth and claws to the attacker , a lack of disproportionate wounds on these sites may simply reflect the success of such defenses . indeed , if the face ( one of the four areas measured ) had a relatively even share of wounds ( west and packer , 2002 ) even though it is much the smallest of the four areas evaluated , this may suggest that faces were wounded disproportionately to their area , perhaps in consequence of a facing defense that served to protect the neck and shoulders . our own observations of adult male lions suggest that facial wounds are very common ( see figure 1 ) . at any rate , the argument that a lion 's mane is protective applies to animals that have manes , i.e. adult males . if such protection were equally important for females and subadult males , and this was indeed the basis on which manes have evolved , then presumably females and subadult males would have manes as well . a second finding relates to approach and avoidance responses of wild lions to dummies set up to have long or short manes , and , light or dark manes . females approached the dark - maned model , and the long - maned model , proportionately more than did males ( west and packer , 2002 ) . female choice of both is consonant with findings that dark - maned males have higher testosterone levels than light - maned males , and that mane length ( and other aspects of quality ) reflect health as well as genetic differences . male avoidance of these particular dummies may well reflect the same factors , and is also consonant with a view that mane quality provides protection against attack , providing abundantly maned lions an advantage in agonistic situations . a particularly interesting factor in the relationship between lion manes and lion social systems is that there are two groups of lions in which males show much less abundant manes ; asian lions in the gir forest of india , and the tsavo lions of lowland kenya . due to human pressures on their habitat , the gir lions are basically a remnant population , albeit with relatively high genetic diversity ( sachdev et al . , 2005 ) but the tsavo lions are doing well , and lack of a mane does not appear to have had any effect upon their reproductive efficiency ( gnoske et al . , 2006 ) , or on their predatory capacity : two of these male lions were notorious for killing over 100 workers on the kenya - uganda railway , at the end of the 19 century ( patterson , 2004 ) . gir lions have substantially scantier manes than those in africa , with the exception of the tsavo region , while the tsavo lions are typically characterized as maneless . abundantly - maned male lions disproportionately overheat when the ambient temperature is high ( west and packer , 2002 ) , strongly suggesting that the relatively poor manes of lions in both areas constitutes a direct evolutionary response to the heat - based maladaptiveness of manes in these local climates . tsavo , lying between the upland plateau of kenya and the coast , is within three degrees of the equator and has no cool months of the year . the gir forest has year - round high levels of both heat and humidity , producing a climate where discomfort from heat and humidity is rated as high or extreme for 10 of the 12 months of the year ( bbc weather service ) . these climate factors , taken in conjunction with direct measures of overheating of lions with manes in hot weather , appear to provide a sufficient and direct explanation for the manelessness of tsavo and gir males . if lion manes arise because they serve as a permissive factor in the context of multi - male prides , what happens to multi - male prides when males do not have manes ? in particular , if mane reduction in these lions is an evolutionary response to long - term high heat and humidity in the environment , alterations in their social systems may be secondary to such male mane changes , i.e. represent an effect of mane changes on sociality . surveying 13 lion groups of known age and sexual composition in tsavo , kays and patterson ( 2002 ) reported that only 2 of the 13 groups had more than one male , and both of these were nomadic , male - only groups . the male - female prides surveyed had an average of more than seven females each , indicating that resource scarcity was not important in group size , and could not account for the presence of only a single male per group . these demographics constitute a very striking difference from lion prides in other areas of africa . schaller ( 1972 ; table ii ) notes that in 14 serengeti lion prides , there were between two and four adult males each , with no pride having only a single adult male . however , in the kays and patterson ( 2002 ) study , mane scores based on length , thickness , and color of hair were determined for males of four prides and three nomad groups . mean scores were somewhat higher for the pride males than for the nomad groups , with the latter containing a higher proportion of young males . however , these scores overlapped completely , ranging from 0 to 24 for the pride males and 0 to 24 for the nomad group males , suggesting that female choice based on male mane characteristics was not a major factor in the composition of prides . while this finding may seem somewhat at variance with west and packer 's ( 2002 ) finding that females tended to approach male dummies with darker / longer manes , it emphasizes that the motivations involved in such approaches are not clear . the appearance of a strange male in a female 's pride territory may well elicit approaches based on motivations other than sexual interest ( e.g. schaller , 1972 , p. 53 ) , including assessment of the risk posed to herself and her offspring . the gir lions show an even more deviant pattern of sociality , compared to those in most areas of africa . in the gir forest .. male and female lions lead separate lives and rarely associate with each other prides are composed of related females , their young , and subadult male offspring ( sunquist and sunquist , 2002 , p. 293 ) . thus in both tsavo and gir , the scantiness or lack of manes in male lions is associated with a particular change in sociality , in which adult males do not simultaneously associate with both females and each other . notably , the difference does not involve a reduction in association with females per se , as the tsavo lion prides typically include one male and several females , nor does it involve a lack of male associations , as both tsavo and gir males often associate in male - only groups . the crucial feature appears to be whether multiple males and females associate in relatively long - term relationships , i.e. prides . in the areas where they occur , smuts ( 1982 ) noted that prides without males in constant attendance failed to raise any cubs . as females are the primary hunters in most prides ( schaller , 1972 ) , this failure to rear cubs probably is not due to cub starvation in prides without males , but instead is the consequence of infanticide by nomadic males ( bertram , 1978 ; packer and pusey , 1983 ) . this suggests that multi - male , mixed - sex lion prides enhance reproductive success by protecting the pride from take - over and subsequent infanticide by other males . if this analysis is valid , the adaptiveness of male manes may be to allow males within prides to fight , while reducing the chance of lethal contact with the particularly vulnerable sites that are normally protected by the mane . there is some difference of opinion ( or observation ) about the levels of aggression by male lions in a courtship context , with some authorities reporting vigorous fighting ( guggisberg , 1961 ) while others ( schaller , 1972 ) do not . male fighting influences access to females in estrus , the male mane would have an obvious adaptive advantage in this context . however , fighting in contexts other than mating can also provide an arena for realization of the adaptiveness of manes in pride males . in particular , lions show little restraint of aggression when feeding , showing few inhibitions about slashing and biting fellow pride members : schaller ( 1972 ; p. 135 ) suggests that this is due to their lack of a true dominance hierarchy , related to the generally solitary life - styles of felids . there is no reason to believe that such disputes are less common , or potentially less damaging , in male male groups . however , if fighting within a nomadic multi - male group is sufficiently damaging as to injure or drive away group members , this has little or no immediate impact on the reproductive success of males in the group , although it might reduce the probability of future success in taking over a pride . in contrast , for a pride male to be injured or driven off makes the pride immediately and substantially more vulnerable , and the infanticide that typically follows a successful take - over may completely eliminate the progeny of the pride males ( packer et al . , 2009 ) . so , to go back to the question posed in the introduction to this article , is it male male fighting , or female choice , or both , that makes the lion 's mane adaptive ? in favor of a view that male male contests are made more adaptive by manes are findings that : ( 1)fatal injuries from lion fights often involve the head and nape.(a)there is some evidence for targeting of these sites.(b)lion fighting involves frontal confrontations such that damage in these areas might be expected in the absence of evolved targeting and(c)these areas are particularly vulnerable , in terms of immediate mortality in response to bites and blows.(2)when manes are reduced , likely by environmental factors , multi - male , mixed - sex prides are vanishingly rare . lion fighting involves frontal confrontations such that damage in these areas might be expected in the absence of evolved targeting and these areas are particularly vulnerable , in terms of immediate mortality in response to bites and blows . when manes are reduced , likely by environmental factors , multi - male , mixed - sex prides are vanishingly rare . the west and packer ( 2002 ) findings do suggest some female choice of more abundantly maned males , in that : ( 1)females approached heavily - maned dummies more than did males.(2)also , field studies indicate that female lions do exercise a substantial degree of mate choice , although it is not known if this reflects male mane characteristics.(3)mane quality is a sensitive index of health in lions . females approached heavily - maned dummies more than did males . also , field studies indicate that female lions do exercise a substantial degree of mate choice , although it is not known if this reflects male mane characteristics . putting these findings together , it seems most like that both is the best answer , but with the further provision that female choice appears to be based on a characteristic that is independently adaptive in terms of the social characteristics of most lions . that is , the mane honestly advertises the quality of a trait that is adaptive for males ( see johnstone , 1995 for a discussion of this view of sexual section ) . indeed , if the mane were selected simply on the basis of female choice , albeit with the result that male offspring with high levels of this trait would similarly prove attractive to females , this would raise a legitimate issue of why only female lions , of all the many felid species , show such a choice . the relationship between multi - male mixed - sex social groups and male manes might conceivably have arisen as a coincidence . however , the coincidence explanation is substantially challenged by findings that mane reductions in the gir and tsavo lions , that appear to be the result of persistent hot or hot and humid environments known to produce overheating in maned lions , are associated with a virtual elimination of multi - male prides . this may reflect nothing more than a human propensity to be intrigued by unique or unusual features of the natural world . the lion 's mane is a highly unusual , sexually dimorphic , male feature , arising about the time of puberty , found in a species that is more social than are its phylogenetic relatives , and in possession of weapons that may be lethally applied to others of its kind : the feature covers an area that is directly accessible in frontal confrontations , may be a particular target of attacks , and is highly vulnerable , as indexed by potential mortality when wounded . morris ( 1968 ) , in the naked ape raised the intriguing question of why humans have less , or shorter and thinner , hair on most body sites . perhaps an even more intriguing question is why humans , absolutely uniquely in the animal kingdom , have two sexually dimorphic hair patches that grow indefinitely . moreover , one of these patches , the beard , begins to grow during puberty , and is thickest in young adulthood and full maturity , tending to decline in old age . like the lion 's mane , male beards are widely assumed to be somewhat adaptive in the context of providing a visual aid to identification of gender at a distance ; in advertising social dominance ; or as sexually attractive to women ( barber , 1995 ) . as evidence for the latter is inconsistent ( feinman and gill , 1977 ; hatfield and sprecher , 1986 ) , and the accuracy and value of other presumed adaptive functions of beards are debatable ( barber , 1995 ) , the near - universal existence of male beards in humans , albeit variable in individual magnitude , remains something of a mystery . figure 1 provides a very good look at the facial and neck structures covered by beards , as well as some estimation of the degree of coverage they may confer . these areas are front and center in aggressive confrontations and may be especially vulnerable to blows when weapons are not used , or to blows from the blunt force weapons that have been available throughout much of hominid evolution . the glass chin phenomenon is well known in boxing , and direct blows to the front and sides of the neck as well as the area just under the nose can be particularly lethal . both are particular targets of attack in unarmed combat techniques ( schillingford , 2001 ) . this protective feature of human beards is also recognized in the technical and competition rules of the international amateur boxing association , which prohibit beards in boxing matches . in addition , based on extensive observations of encounters in young children , the child psychologist margaret manning has suggested ( personal communication ) that the head / face / neck area is a specific target of attack in young children . this is particularly interesting as combat training is unlikely to be involved in childrens preferences , and it may provide something of a functional or adaptive basis for the nonsexually dimorphic , evergrowing hair patch that covers the dorsal , side , and back sections of the head . notably , if left to grow , head hair also covers the back and sides of the neck , leaving the front of the neck , the most vulnerable aspect in frontal confrontations , exposed except in pubertal and postpubertal males . the many points where manes and beards show parallels also bring up the interesting question of why other mammalian species with potentially lethal weaponry fail to develop such protections . one answer , already mentioned , is that they may be much less necessary in species with solitary life - styles . another is that a structure such as the horns or antlers of ungulates may simultaneously serve as weapon , target site , and protection against this specialized form of intraspecific attack . indeed , such protective weaponry is much more common in gregarious than in solitary species of african bovids ( estes , 1992 ) . however , many other social species have weapons that are clearly capable of causing lethal conspecific damage , but have no notable structures to protect highly vulnerable sites . one possible reason for this omission may be that animals with both dangerous weapons and a long and consistent evolutionary history of sociality , such as most canids , tend to have a clearer within - group dominance structure than do lions , including behavior mechanisms ( submission ) that reduce intraspecific attack ( macdonald and sillero - zubiri , 2004 ) . humans do certainly have both dominance hierarchies and submissive behaviors , but these appear to vary widely across cultures and their existence and extent in precultural humans is difficult to estimate . while there appears to be a linear dominance hierarchy in male chimpanzees ( goldberg and wrangham , 1997 ) , this appears not to be so in bonobos ( paoli et al . , 2006 ) ; these having equal claim to the ancestral line from which humans split . perhaps lions and humans came to their need for protection against lethal intraspecific attack through slightly different routes ; lions with long - term lethal weapons but lately evolving a social life - style , whereas people are from a line with substantial , though not clearly delineated , sociality , but lately evolving the development and use of lethal weapons . both species may have encountered the combination of a social life - style and lethal weaponry too rapidly to evolve innately - organized social mechanisms that protect individuals from the damaging consequences of intra - group attack . this view implies that structural protections ; here , long , coarse , and abundant hair growth in relevant sites , may be more easily or rapidly evolved than are complex behavioral changes . however , it is only one of many potential questions following from the present analysis . it seems likely that a combination of experimental and ethological approaches may be necessary in order to gain a better understanding of the complex evolutionary interplay between sociality and other behavioral and structural characteristics of particular species . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . proteomic studies typically use tandem mass spectrometry ( ms / ms ) employing collision - induced dissociation ( cid ) for identification ( and quantification ) of large numbers of peptides and proteins [ 1 , 2 ] . however , complete assignment of the product ions remains impossible because of limitations in our understanding of peptide dissociation pathways . low energy cid typically results in charge - directed cleavage of amide bonds in accordance with the mobile proton model [ 3 , 4 ] , which describes the intramolecular transfer of a proton from a basic moiety to a heteroatom along the peptide backbone . weakening of the peptide bond results and fragmentation ensues , generating diagnostic b- , y- , and a - ions . these product ions are then used to determine the amino acid sequences of the precursor peptides and hence elucidate identity of the proteins whence they derive . the structure of c - terminal y - ions is recognized to be that of a truncated peptide ( yn ) or protonated amino acid ( y1 ) . however , the structure of their b - ion counterparts is still the subject of much interest . more recently , b - ions have been shown to incorporate a c - terminal oxazolone ring . nucleophilic attack by the n - terminal amine on the electrophilic carbonyl carbon within this protonated oxazolone ring results in the formation of a fully macrocyclic intermediate from the linear oxazolone - terminating b - ion [ 68 ] , with the prevalence of macrocycle formation increasing with b - ion length [ 912 ] . we have previously demonstrated that cid of protonated linear yagfl - nh2 results in the formation of a heterogeneous b5 ion population with respect to drift time during analysis by traveling - wave ion mobility spectrometry - ms . this observation is consistent with the formation of a macrocyclic intermediate that is stable over the timescale of the ion mobility ( i m ) separation . many other more recent studies utilizing im - ms , infrared multiphoton dissociation ( irmpd ) spectroscopy , electron - transfer dissociation ( etd ) , hydrogen / deuterium ( h / d ) exchange , and theoretical calculations , often in combination , support the observation of multiple b - ion conformations / isomers , arising during cid [ 8 , 9 , 1418 ] . for example , direct spectroscopic evidence for the existence of a macrocyclic intermediate has been obtained from irmpd analysis of the b4 ion of leu - enkephalin and the b5 ion produced from g5r . once formed , this macrocyclic intermediate can ring open at various amide bonds , yielding non - native or scrambled peptide sequences , where previously internal amino acid residues become exposed at the termini . these rearranged species can then undergo secondary fragmentation events , which are particularly prevalent following cid using a qtof type collision cell , producing a product ion series not representative of the initial peptide sequence . macrocycle formation thus functions as a precursor to the formation of non - native , or scrambled , product ions . use of the common peptide fragmentation nomenclature [ 21 , 22 ] to describe such scrambled sequence ions is fraught with confusion ; consequently , we have recently proposed an extension to this nomenclature , enabling secondary product ions resulting from macrocycle formation to be easily assigned . a study by harrison showed that the abundance of product ions generated from these non - native sequences increases with elevated cid collision energies . these experiments and others ( e.g. , ) also demonstrate that sequence scrambling can be prevented through acetylation of the peptide n - terminus , a finding that supports the proposed mechanism of macrocycle intermediate formation . of concern when considering the propensity for occurrence of such complex fragmentation pathways is that facile sequence rearrangement and subsequent fragmentation can result in the production of abundant scrambled product ions and , hence , misassignment of the original peptide sequence . research into the frequency with which such scrambled sequences are observed and the potential consequences of this rearrangement for large scale ms / ms analyses has been conducted [ 12 , 25 , 26 ] . however , there remains a dearth of understanding regarding the extent to which such sequence scrambling occurs or , indeed , the multitude of factors which potentially influence the process . the majority of studies aimed at facilitating understanding of these rearrangement mechanisms primarily utilize synthetically generated conservative a number of investigations have been conducted to assess the effect of peptide size on sequence scrambling ; van stipdonk and coworkers reported that for the tetrapeptide yafg and permuted isomers , cid of the b3 ion does not result in the production of non - sequence ions , an observation rationalized by considering the extensive steric strain that would result following cyclization . however , they did observe product ions indicative of sequence scrambling for larger species , ranging from pentapeptides to decapeptide methyl esters . the increased size and associated degrees of freedom of these larger species may be expected to disfavor macrocycle formation , but the results are in accordance with our own observation for the scrambling of the b9 ion produced from the peptide neurokinin dmhdffvglm - nh2 . tirado and polfer have recently conducted a more extensive investigation to better understand the effect of b - ion length in the prevalence of macrocycle formation . however , size alone does not account for all the b - ion structures observed , with subtle differences in the sequence also playing a role . although small b2-ions generally prefer to adopt oxazolone structures , the inclusion of basic residues ( e.g. , histidine , arginine ) has been demonstrated to result in enhanced formation of diketopiperazine structures [ 29 , 30 ] . macrocycle formation of larger b - ions also appears to be disfavored in the present of proline , resulting preferentially in oxazolones . atik and yalcin conducted a detailed study to determine the effect of acidic side chains on peptide rearrangement pathways , but observed little dependence on the extent of sequence scrambling on the position of glutamic acid and aspartic acid side chains within the peptide structure . conversely , it has been reported by bythell et al . that the position of the basic amino acid histidine within permuted isomers of an alanine - rich hexapeptide sequence does influence the observed scrambling of the b5 ion generated upon cid . location of the histidine residue near the peptide n - terminus precluded cyclization , yet situation of this basic residue near the c - terminus of the initial b5 ion permitted observation of the scrambled products . the presence of arginine and lysine residues can also heavily influence observed peptide decomposition pathways , either because of the formation of salt bridging interactions [ 34 , 35 ] or through nucleophilic attacks [ 3537 ] . these factors might , therefore , be expected to influence the formation and subsequent ring opening of the b - ion macrocycle . interestingly , in contrast to other amino acids with nucleophilic side chains , the presence of an internal arginine residue appears to inhibit peptide scrambling . this effect has been attributed to the high gas - phase basicity of the arginine side chain , which sequesters the available proton , thereby preventing facile macrocycle formation . however , lack of formation of the macrocycle precursor is not the sole explanation for why a low abundance of ions derived from sequence scrambling may be observed from a particular precursor . two other possibilities exist , namely , failure for the macrocycle to ring open following formation or failure of the ring - opened linear intermediate to fragment further . in our present study , we address these possibilities through analysis of a selection of peptides produced by proteolysis of either bovine serum albumin ( bsa ) or enolase ( s. cerevisiae ) , using the proteases lys - n ( cleaving n - terminal to lysine residues ) and lys - c ( cleaving c - terminal to lysine residues ) , and demonstrate differences in the extent of scrambled product ions generated for some of the resultant peptides . formation of the more highly basic homoarginine residue by guanidination of n - terminal lysine residues was subsequently used to help elucidate the role of gas - phase basicity in peptide rearrangement . the proteins bovine serum albumin ( bsa ) and enolase from s. cerevisiae were obtained from sigma aldrich ( poole , uk ) and resuspended in 50 mm ammonium bicarbonate ( 10 pmol/l ) . each was then reduced with dithiothreitol ( 4 mm , 60c , 45 min ) and alkylated ( iodoacetamide , 14 mm , room temperature , dark , 1 h ) . the dtt concentration was increased to 7 mm and samples proteolyzed overnight with lys - n or lys - c proteases ( 2% [ w / w ] , 37c ) . an aliquot ( 100 pmol ) of each of the peptide digestion mixtures was dried by vacuum centrifugation and reconstituted in 10 l h2o . to this , ammonium hydroxide ( 7 m , 10 l ) and o - methylisourea ( 0.5 m in h2o , 5 l ) were added and the reaction mixtures left overnight at room temperature . each of the derivatized samples were again dried by vacuum centrifugation and reconstituted in 10 l h2o , prior to desalting using c18 ziptips ( millipore , watford , uk ) . peptide mixtures ( both derivatized and underivatized ) were diluted to a final concentration of 1 pmol/l using 50% ( v / v ) acetonitrile in water containing 0.1% ( v / v ) formic acid , and infused into a synapt hdms instrument ( waters , manchester , uk ) using gold - coated nanospray emitter tips ( proxeon , odense , denmark ) . the capillary voltage , cone voltage , and source temperature were set at 2.3 kv , 40 v , and 80c , respectively . the m / z of interest was mass selected by the quadrupole mass analyzer prior to being subjected to cid in the trap collision cell . the collision energy applied in this region was manually tuned to give optimal peptide fragmentation , such that the precursor ion abundance was ~10% that of the base peak . further analysis of the peptide fgeralk ( 1 pmol/l in 50% ( v / v ) acetonitrile in water containing 0.1% ( v / v ) formic acid was conducted by direct infusion nano - electrospray ionization ( nesi ) ( 5 l / min ) using an amazon ion trap ( bruker , bremen , germany ) . mass selection at m / z 410.7 corresponding to that of the peptide of interest was performed , followed by cid with the fragmentation amplitude set to 0.35 ev , with data collected between m / z 150 and 1800 . the proteins bovine serum albumin ( bsa ) and enolase from s. cerevisiae were obtained from sigma aldrich ( poole , uk ) and resuspended in 50 mm ammonium bicarbonate ( 10 pmol/l ) . each was then reduced with dithiothreitol ( 4 mm , 60c , 45 min ) and alkylated ( iodoacetamide , 14 mm , room temperature , dark , 1 h ) . the dtt concentration was increased to 7 mm and samples proteolyzed overnight with lys - n or lys - c proteases ( 2% [ w / w ] , 37c ) . an aliquot ( 100 pmol ) of each of the peptide digestion mixtures was dried by vacuum centrifugation and reconstituted in 10 l h2o . to this , ammonium hydroxide ( 7 m , 10 l ) and o - methylisourea ( 0.5 m in h2o , 5 l ) were added and the reaction mixtures left overnight at room temperature . each of the derivatized samples were again dried by vacuum centrifugation and reconstituted in 10 l h2o , prior to desalting using c18 ziptips ( millipore , watford , uk ) . peptide mixtures ( both derivatized and underivatized ) were diluted to a final concentration of 1 pmol/l using 50% ( v / v ) acetonitrile in water containing 0.1% ( v / v ) formic acid , and infused into a synapt hdms instrument ( waters , manchester , uk ) using gold - coated nanospray emitter tips ( proxeon , odense , denmark ) . the capillary voltage , cone voltage , and source temperature were set at 2.3 kv , 40 v , and 80c , respectively . the m / z of interest was mass selected by the quadrupole mass analyzer prior to being subjected to cid in the trap collision cell . the collision energy applied in this region was manually tuned to give optimal peptide fragmentation , such that the precursor ion abundance was ~10% that of the base peak . further analysis of the peptide fgeralk ( 1 pmol/l in 50% ( v / v ) acetonitrile in water containing 0.1% ( v / v ) formic acid was conducted by direct infusion nano - electrospray ionization ( nesi ) ( 5 l / min ) using an amazon ion trap ( bruker , bremen , germany ) . mass selection at m / z 410.7 corresponding to that of the peptide of interest was performed , followed by cid with the fragmentation amplitude set to 0.35 ev , with data collected between m / z 150 and 1800 . to study the effect of increasing the gas - phase basicity of the n - terminal residue on sequence scrambling , it was first necessary to identify those peptides produced by digestion with lys - n that exhibited evidence of significant rearrangement during cid . a selection of native lys - n peptides and their corresponding derivatized sequence , now with an n - terminal homoarginine residue , were isolated and subjected to cid in the trap collision cell of a synapt hdms instrument ( waters ) . as expected , increased collision energy was required to induce dissociation of the derivatized analogue because of reduced mobility of the proton now sequestered by the homoarginine residue . figure 1 compares the cid product ion spectra produced from analysis of the native and lysine - derivatized forms of the singly protonated peptide kgvlhav . the spectrum derived from the native peptide ion ( figure 1a ) contains several species attributable to scrambling of the original primary sequence . however , these rearranged product ions are not observed in the ms / ms spectrum of the derivatized peptide ( figure 1b ) . the b6 ion of the derivatized peptide ( k*gvlha ) does not appear to be generated during cid and , hence , the precursor to formation of many of the observed rearranged product ions is not present . however , there is an abundant peak at m / z 666.40 that corresponds to the b6+h2o ion ; such species have previously been shown by thorne et al . to have a linear structure analogous to that of an intact peptide . as the oxazolone moiety is required for the macrocyclic intermediate to form , scrambled species can not , consequently , be generated from this linear ion . moreover , the [ b53]b4 scrambled species in the native peptide product ion mass spectrum derived following rearrangement of the primary b5 product ion ( labeled according to the chawner et al . these differences can only be explained by the induced change in the n - terminal residue side chain . curiously , a number of product ions were observed from these lys - n - generated peptides that could not be assigned after consideration of either linear or b - ion rearranged product ions , and their likely identity has yet to be determined . the observation of unassigned product ions is in contrast to the typical ion trap cid spectra of lys - n peptides exemplified in taouatas et al . , but apparent in the few qtof cid spectra presented by rao et al . , suggesting that these ions may in part be an effect of the n - terminal basic residue on secondary fragmentation pathways occurring during beam - type cid.figure 1comparison of qtof ms / ms spectra resulting from ( a ) cid of [ m+h ] peptide kgvlhav at m / z 723.45 , and ( b ) cid of [ m+h ] peptide k(guanidinyl)gvlhav at m / z 765.47 comparison of qtof ms / ms spectra resulting from ( a ) cid of [ m+h ] peptide kgvlhav at m / z 723.45 , and ( b ) cid of [ m+h ] peptide k(guanidinyl)gvlhav at m / z 765.47 further examples of the enhanced formation of b(n-1)+h2o species following guanidination of the n - terminal lysine residue are shown in knvply ( figure 2a ) and klvtdlt ( figure 3a ) ; both decompose during cid to produce minor b(n-1)+h2o species corresponding to loss of the c - terminal amino acid , in addition to generating ion series that are indicative of sequence rearrangement . in each example following derivatization , the peptide ion containing the n - terminal homoarginine residue dissociates to produce a significantly more abundant b(n-1)+h2o species , with no sequence scrambled species being observed ( figures 2b and 3b ) . in the case of knvply ( figure 2 ) , the native peptide generates scrambled products from b5 ( including a pseudo c- terminal product ion designated [ b52]y3 ) , whereas the derivatized peptide yields the b5+h2o ( and b4+h2o ) product ion upon cid . likewise for klvtdlt ( figure 3 ) , formation of the b6+h2o and b5+h2o product ions upon n - terminal derivatization appear to preclude the generation of b5- and b6-derived scrambled products.figure 2comparison of qtof ms / ms spectra resulting from ( a ) cid of [ m+h ] knvply at m / z 733.42 , and ( b ) cid of [ m+h ] derivatized peptide k(guanidinyl)nvply at m / z 775.45 . only those scrambled species with significant abundance ( ~10% ) are labeled ; y represents a product ion arising the pseudo c - terminal portion of the rearranged b - ionfigure 3comparison of qtof ms / ms spectra resulting from ( a ) cid of [ m+h ] klvtdlt at m / z 789.47 , and ( b ) cid of [ m+h ] derivatized peptide k(guanidinyl)lvtdlt at m / z 831.49 . although a large number of scrambled species were present ( a ) , only those of significant abundance are labeled comparison of qtof ms / ms spectra resulting from ( a ) cid of [ m+h ] knvply at m / z 733.42 , and ( b ) cid of [ m+h ] derivatized peptide k(guanidinyl)nvply at m / z 775.45 . only those scrambled species with significant abundance ( ~10% ) are labeled ; y represents a product ion arising the pseudo c - terminal portion of the rearranged b - ion comparison of qtof ms / ms spectra resulting from ( a ) cid of [ m+h ] klvtdlt at m / z 789.47 , and ( b ) cid of [ m+h ] derivatized peptide k(guanidinyl)lvtdlt at m / z 831.49 . although a large number of scrambled species were present ( a ) , only those of significant abundance are labeled observation of enhanced b(n-1)+h2o ion formation is consistent with the findings reported by various groups [ 40 , 43 , 44 ] ; the c - terminal rearrangement required for the formation of such ions is promoted by the presence of a basic residue at the n - terminus . derivatization of the n - terminal lysine side chain to homoarginine enhances the gas - phase basicity of this residue , increasing the extent of rearrangement and thus permitting b(n-1)+h2o ion formation . the apparent inhibition of peptide sequence scrambling following derivatization may , therefore , simply be a result of ion current being directed away from the formation of oxazolone - terminated b - product ions , reducing the abundance of the precursors to sequence scrambled species . alternatively , the reduced complexity of the observed products of fragmentation following derivatization may result because of the presence of a homoarginine residue , analogous to the observation reported by molesworth and van stipdonk for arginine containing sequences . molesworth and van stipdonk concluded that inhibition of sequence scrambling for such peptides may be the result of either a reduced propensity for macrocycle formation via rearrangement of the initially formed oxazolone structure , or the prevention of direct generation of a cyclic b - ion structure on cid of the precursor ion . sequester a proton necessary for facile macrocycle formation or , alternatively , prevent head to tail cyclization of linear , oxazolone - terminated b - ions by permitting the formation of a structure stabilized by an intramolecular hydrogen bonding interaction . if such factors influence the prevalence of sequence scrambling , it may be expected that a similar effect would be observed following modification of the lysine side chain to homoarginine . consequently , it is possible that the inhibition of sequence scrambling may arise simply as a result of the absence of a precursor to macrocycle formation . however , the observation of b6 and b5 product ions from klvtdlt both prior to and following derivatization suggests that the homoarginine residue does indeed play a role in the inhibition of sequence rearrangement . to further test this hypothesis , the peptide sequences rgvlhav , rnvply , and rlvtdlt were synthesized ( jpt peptide technologies , berlin , germany ) and subjected to equivalent analysis by cid . the resultant product ion mass spectra are shown in figure 4 . as observed previously , extensive formation of b(n-1)+h2o species can be detected for each of these peptides and no product ions resulting from sequence scrambling are present . thus , it would appear that the presence of an arginine or homoarginine residue precludes sequence scrambling and the highly basic n - terminal residue promotes rearrangement at the c - terminus to give b(n-1)+h2o product ions.figure 4comparison of qtof ms / ms spectra resulting from ( a ) cid of [ m+h ] rgvlhav at m / z 751.45 , ( b ) cid of [ m+h ] rnvply at m / z 761.43 , and ( c ) cid of [ m+h ] rlvtdlt at m / z 817.47 comparison of qtof ms / ms spectra resulting from ( a ) cid of [ m+h ] rgvlhav at m / z 751.45 , ( b ) cid of [ m+h ] rnvply at m / z 761.43 , and ( c ) cid of [ m+h ] rlvtdlt at m / z 817.47 as mobilization of the proton sequestered by the arginine side chain would normally be required for efficient cid , molesworth et al . instead proposed mobilization of an amide proton from a leucine residue , leading to b5 ion formation , in accordance with the model previously published by paizs and coworkers . this proposed fragmentation pathway proceeds via an iminol tautomer to generate a b5 ion following nucleophilic attack to yield a deprotonated oxazolone ring . each of the sequences in our present study also incorporates a leucine residue and , therefore , may potentially fragment via the same pathway once the mobile proton is sequestered by the homoarginine residue . to help determine whether inhibition of peptide scrambling for arginine ( or homoarginine ) containing sequences is related to the lack of a proton that can be mobilized upon activation , the doubly protonated peptide fgeralk was subjected to cid . figure 5a shows the product ion mass spectrum generated from this peptide using a qtof mass spectrometer , with an abundant ion observed at m / z 545.34 ( [ b63]b5 ) whose origin is attributable to ring opening of an intermediate macrocycle structure . the abundance with which this product ion is observed is somewhat surprising as scrambled species are typically minor components of ms / ms spectra . the mechanism by which this scrambled product ion is generated will be determined by the immediate precursor to the rearrangement process . the doubly charged , even electron precursor ion either fragments to give a singly charged ion pair originating from opposing termini of the peptide ( a singly charged b - ion and a singly charged y - ion ) or a doubly charged a6 fragment resulting from retention of two protons within the n - terminal product ion series.figure 5(a ) qtof ms / ms spectrum generated by cid of the [ m+2h ] peptide fgeralk . ( b ) quadrupole ion trap ( qit ) ms / ms spectrum generated by cid of the [ m+2h ] peptide fgeralk ( a ) qtof ms / ms spectrum generated by cid of the [ m+2h ] peptide fgeralk . ( b ) quadrupole ion trap ( qit ) ms / ms spectrum generated by cid of the [ m+2h ] peptide fgeralk as it is unlikely that the a6 product ion could fragment further to generate an ion of m / z 545.34 ( equivalent to [ b63]b5 ) , we hypothesize that ring opening of the macrocycle occurs preferentially c - terminal to glutamic acid promoted by the acidic side chain , as is frequently observed during cid of linear peptide ions ( scheme 1 ) . observation of the [ b63]b5 ion at such a level suggests that its formation is attributable to ring opening of the macrocycle intermediate between the e and r residues ( position 3 ) , generating the scrambled product ralfge , followed by subsequent loss of the now c - terminal glutamic acid residue . additional scrambling pathways in the qtof spectrum produced from fgeralk are not present at a detectable level , except for a [ b53]b4 product ion observed at m / z 432.24 , corresponding to the same charge remote cleavage of a macrocyclic intermediate , this time arising from the b5 product ion . if ring opening were initiated by a mobile proton , a more heterogeneous population of sequence scrambled product ions would be expected , with ring opening occurring at multiple sites . equivalent cid analysis of the fgeralk peptide using an amazon ion trap ( bruker , bremen , germany ) provides evidence to support this suggested fragmentation pathway ( figure 5b ) . the relative abundance of the [ b63]b5 product ion with respect to the y4 ion is much reduced in the qit spectrum in comparison to that observed following cid in the qtof . the y4 ion was selected for internal reference as the yield of this product ion should be relatively consistent under the conditions encountered during cid on the different instrument platforms . further inspection of the relative abundances of the peaks associated with the overlapping isotope patterns of the b6 ( m / z 674.36 ) and y6 ( m / z 673.39 ) ions in each spectrum shows that the b6 population is depleted during analysis in the multiple collision regime of the qtof collision cell ( figure 6a and b ) , whereas the singly charged a6 ion ( figure 5 ) is observed with significant abundance during each analysis . it has previously been demonstrated that n - terminal product ions are less stable toward secondary fragmentation events than their y - ion counterparts under such conditions and as a consequence are often under - represented in qtof ms / ms spectra compared with those obtained using a qit . during cid on a qtof instrument , peptide ions are subject to multiple collisions with the neutral buffer gas causing preferential secondary decomposition of b - ion species to lower members of the ion series . in this instance , it would appear that during analysis on the qtof platform , once initial decomposition has occurred , the b6 and b5 product ions undergo further activation , promoting rearrangement and secondary fragmentation leading to formation of the [ b63]b5 and [ b53]b4 species ; there is also an increase in the relative abundance of the smaller b4 ( and b5 ) ion(s ) , as might be expected following multiple dissociation events from larger species ( figure 5 ) . this observation is highlighted by analysis of the y6 ion isotope ratio observed from analysis on the qtof instrument at high and low collision energy . at lower collision energy ( figure 6a , collision energy set at 19.5 ev ) there is a distortion of the distribution of ion current , suggesting a contribution to this isotopic envelope from the b6 ion.scheme 1formation of the intense [ b63]b5 product ion following cid of the [ m+2h ] peptide fgeralk is dependent on glutamic acid - mediated cleavage of the b6 macrocyclefigure 6cid of [ m+2h ] fgeralk with the collision energy set at either ( a ) 19.5 ev or ( b ) 26 ev using a qtof instrument results in formation of a y6 ion at m / z 673.39 . distortion of the isotope distribution in ( a ) suggests a contribution from the b6 ion at m / z 674.36 . shown in the inset is the theoretical isotope distribution of the b6 ion which was modeled using the ms - isotope program within protein prospector . ( c ) equivalent cid analysis of [ m+2h ] fgeralk using a qit instrument results in formation of an abundant b6 ion at m / z 674.36 . slight calibration differences between the qtof and qit instruments used for analysis means there is a small discrepancy in the observed m / z . conversely , at higher collision energy ( figure 6b , collision energy set at 26 ev ) , there is no distortion of the isotope distribution , reflecting depletion of the b6 ion population through secondary fragmentation and formation of the [ b63]b5 scrambled product ion . this phenomenon is of reduced consequence during qit analysis as products of initial fragmentation are no longer resonant with the excitation voltage and are , therefore , not subject to multiple ion - neutral collisions . higher members of the b - ion series are thus often observed , and this is demonstrated in figure 6c where , in contrast to the qtof data , the b6 ion generated from fgeralk is observed with greater abundance than the y6 ion . generation of the scrambled [ b63]b5 species on the qit instrument may be expected to be enhanced by the longer trapping times associated with such analysis . however , it appears that as further activation of the b6/b5 product ions is avoided , so too is rearrangement of the peptide sequence . the a5 ion , although relatively abundant in the qtof data , was not present in the qit tandem mass spectrum . this observation is in line with the reduced population of b5 in the qit subsequently failing to yield adequately detectable levels of a5 as a result of elimination of co. as an aside , it is also interesting to note that the relative abundance of the y - ion series for this peptide differs following cid under the qit or qtof regimes , with y4>y5>y6 on the qit , whereas this order is reversed on the qtof . previous comparisons of y - ion abundance between qit and qtof instruments have failed to discern any significant bias , and we are currently at a loss to rationalize this observation . formation of the intense [ b63]b5 product ion following cid of the [ m+2h ] peptide fgeralk is dependent on glutamic acid - mediated cleavage of the b6 macrocycle cid of [ m+2h ] fgeralk with the collision energy set at either ( a ) 19.5 ev or ( b ) 26 ev using a qtof instrument results in formation of a y6 ion at m / z 673.39 . distortion of the isotope distribution in ( a ) suggests a contribution from the b6 ion at m / z 674.36 . shown in the inset is the theoretical isotope distribution of the b6 ion which was modeled using the ms - isotope program within protein prospector . ( c ) equivalent cid analysis of [ m+2h ] fgeralk using a qit instrument results in formation of an abundant b6 ion at m / z 674.36 . slight calibration differences between the qtof and qit instruments used for analysis means there is a small discrepancy in the observed m / z . tandem ms analysis has been used to demonstrate that increasing the gas - phase basicity of n - terminal basic groups through derivatization of the lysine side chain to give homoarginine results in enhanced formation of b(n-1)+h2o species via a c - terminal rearrangement . a subsequent reduction in the complexity of the products of cid is observed , with no fragments arising from scrambling of the initial peptide sequence detected . this apparent inhibition of scrambling for singly protonated peptides may simply be the result of ion current being directed away from standard b - ion fragments , which are the precursors to macrocycle formation , limiting the production ( and therefore detection ) of sequence scrambled species . however , further analysis of a doubly protonated arginine - containing peptide results in the observation of a product of sequence scrambling upon cid of the precursor ion . highly selective ring - opening of a macrocycle intermediate is promoted locally by the acidic side chain of a glutamic acid residue to give sequence rearrangement via a single decomposition pathway . observation of this highly selective fragmentation from a doubly charged precursor shows that formation of a macrocyclic intermediate ( from a singly protonated b - ion ) can occur even in the presence of a guanidine moiety . this suggests that the common depletion of rearrangement ion products in the presence of a strongly basic residue may be associated with one of two effects , namely ( 1 ) the diversion of n - terminal product ion current to b(n-1)+h2o species , depleting the population of macrocycle precursors , and ( 2 ) the disfavoring of macrocyclic cleavage by the presence of a proton - sequestering site , unless the presence of a glutamic acid residue promotes cleavage by local proton transfer . the presence of an arginine / homoarginine side chain within a peptide sequence will , therefore , in most cases preclude the formation of scrambled product ions , in accordance with previously published studies [ 38 , 49 ] . although the protease lys - n is not currently used extensively in ms - based proteomics , it is slowly gaining in popularity , in part because of its ability to bias fragmentation pathways towards n - terminally derived species and the beneficial effect that this has on electron - transfer - mediated ladder sequencing of peptides . the data presented here demonstrates that peptides with an n - terminal lysine residue are subject to relatively high levels of sequence rearrangement ( peptide scrambling ) during qtof - based cid . indeed , we have failed to account for a proportion of the product ions based on our current understanding of the dissociation mechanisms . we thus suggest that lys - n - generated peptides should be subject to routine guanidination prior to qtof analysis . alternatively , an arg - n protease ( that cleaves n - terminal to arginine residues ) would yield the same results , should one become available . not only do such peptides reduce the complexity of the cid spectrum for annotation ( automated or otherwise ) , but the resultant homoarginine - terminating peptides yield highly diagnostic b(n-1/n-2)+h2o ions of significant abundance . these two features together will improve confidence in sequence identity . exploitation of this highly efficient guanidination reaction will also likely benefit studies employing an orbitrap - based hcd regime and has already proven beneficial during electron - transfer dissociation , improving peptide identification .

the function of manes in lions has been a topic of scientific interest since darwin ( 1871 ) suggested that it provides protection in intraspecific fights . recent experimental studies on wild lions have emphasized the role of female selection , but analyses of specific attack behaviors and targets , and the social consequences of manelessness for lions living in very hot climates suggest that male manes may indeed mitigate the outcomes of intraspecific male attack and thus serve a permissive function for multi - male + female groups , facilitating protection of prides against take - overs and infanticide by nomadic males . humans also have unusual structural protections for the head , face and neck , areas that are especially accessible during intraspecies attack , and highly vulnerable to damage . one of these , the beard , consists of coarse hairs that grow indefinitely , but only for males , and only during and following puberty ; suggesting that it , like the lion 's mane , may serve as protection in intraspecies male fights . such structural protections may reflect a specific combination of lethal weaponry and social life - style , particularly when these are developed so rapidly that they are not accompanied by the evolution of complex attack - inhibiting social behaviors . bottom up proteomic studies typically use tandem mass spectrometry data to infer peptide ion sequence , enabling identification of the protein whence they derive . the majority of such studies employ collision - induced dissociation ( cid ) to induce fragmentation of the peptide structure giving diagnostic b- , y- , and a- ions . recently , rearrangement processes that result in scrambling of the original peptide sequence during cid have been reported for these ions . such processes have the potential to adversely affect ion accounting ( and thus scores from automated search algorithms ) in tandem mass spectra , and in extreme cases could lead to false peptide identification . here , analysis of peptide species produced by lys - n proteolysis of standard proteins is performed and sequences that exhibit such rearrangement processes identified . the effect of increasing the gas - phase basicity of the n - terminal lysine residue through derivatization to homoarginine toward such sequence scrambling is then assessed . the presence of a highly basic homoarginine ( or arginine ) residue at the n - terminus is found to disfavor / inhibit sequence scrambling with a coincident increase in the formation of b(n-1)+h2o product ions . finally , further analysis of a sequence produced by lys - c proteolysis provides evidence toward a potential mechanism for the apparent inhibition of sequence scrambling during resonance excitation cid.graphical abstract

neuronal ceroid lipofuscinoses ( ncls ) are a group of progressive neurogenetic disorders . in 1998 , categorized the ncls into five major groups based primarily on age , clinical presentation , histological findings , and genetic loci ; infantile ncl ( incl ) , late infantile / early childhood ncl ( lincl ) , juvenile ncl ( jncl ) , adult ncl and variant , lincl ( vlincl ) . this classification is still widely used . within the last decade , mutations that cause ncl have been found in 14 human genes ( cln1 , cln2 , cln3 , cln4/dnajc5 , cln5 , cln6 , cln7/mfsd8 , cln8 , cln10/ctsd , cln11/grn , cln12/atp13a2 , cln13/ctsf , cln14/kctd7 , clcn6 , and sgsh ) . infantile ncl with cln1 mutation presents with motor disturbances ( ataxia , hypotonia , spasticity ) , visual impairment , seizures , dementia , and familial occurrence . late infantile with cln2 mutation manifests with onset between 2 and 4 years and symptoms of epilepsy , cognitive decline , ataxia , visual failure , and death in between 9 and 18 years . juvenile ncl , with cln3 mutation presents with progressive visual loss , speech and cognitive decline , epilepsy , behavioral disturbances and death in the 3 or 4 decade . however , they are already reclassified on the basis of newer molecular findings , which have provided a far more overlap for the different genetic variants that have previously suggested by the clinical phenotypes . we report three cases with atypical jncl and pertinent issues concerning diagnostic difficulties , ophthalmologic , neuroradiological , and laboratory findings are discussed . a 9-year - old pakistani boy born to non - consanguineous parents with a normal perinatal period and early development and at 4 years , he began to show regression in motor and cognitive skills . he was unsteady and needed support , when walking at 6 and became wheelchair bound at 7 years . he developed progressive visual loss and was unable to follow a bright red object at 7 years . ophthalmological examination revealed dystrophic appearance of retina , attenuation of retinal vessels with a pale disc . serial cranial magnetic resonance imaging ( mri ) showed diffuse white matter hyperintensity in t2-weighted but not in flair at 5 years and subsequently , cerebral and cerebellar atrophy at 7 years . electroretinogram ( erg ) was absent and visual evoked potential ( vep ) was normal . palmitoyl protein thioesterase ( ppt ) and tripeptidyl peptidase i ( tpp 1 ) enzyme activities were within normal range . skin and muscle biopsy for inclusion bodies were negative and blood film showed no vacuolated lymphocytes . however , ultra structural examination of lymphocytes showed rare , membrane bound , condensed storage material with fingerprint profiles . cln5 gene mutation was positive . at last follow - up at 11 years , he was wheelchair bound but could walk with support , had severe visual impairment and slow and slurred speech . he was the 6-year - old brother of case 1 and his early developments were normal except for speech . he babbled at 5 months , copied noises at 8 months and only spoke with recognizable word at 2 year 7 months ( his brother spoke at 18 months ) . he would play football ( his eldest brother never ran or jumped ) with other children and his expressive language had never been clear and had difficulty putting words together and had developed stammer . he was slow to learn and at 4 years , he began to have cognitive and motor regression . cranial mri findings were very similar to that demonstrated on his brother 's scan . at last follow - up ( 8 years ) , he had drooling saliva , he was wheel chair bound with deterioration in his speech , cognition , motor skills , and fully dependent for his needs . an 11-year-10-month - old caucasian boy with normal perinatal and neurodevelopmental history was referred to us with 3-year history of progressive visual loss and visual hallucinations . his symptoms started with difficulty in reading , worsening handwriting , and flashes of lights in both eyes ( be ) . he gave us a description and consistently drew figures , which he appeared to be seeing in front of his eyes . the figures had a red border with circles and lines criss - crossing the circles . the va at first presentation was 6/60 in be when he was 10 years . at last follow - up at 13 , va was 6/60 in right eye ( re ) and 3/60 in left eye ( le ) . however , electron microscopy ( em ) of buffy coat showed rare , membrane bound , electron dense , granular material associated with a lipid droplet in the cytoplasm of lymphocytes . ppt activity was low measuring 2.2 nmol / hr / mg protein and normal tpp - i . molecular genetic analysis of exon 1 - 9 of cln1 gene showed that he was heterozygote for p.cys96tyr and p.arg151x mutations . a diagnosis of milder form of incl ( cln1 ) was made . at his last neurology follow - up at 13 years he had developed behavioral and cognitive problems . he had not developed seizures or motor difficulties . a 9-year - old pakistani boy born to non - consanguineous parents with a normal perinatal period and early development and at 4 years , he began to show regression in motor and cognitive skills . he was unsteady and needed support , when walking at 6 and became wheelchair bound at 7 years . he developed progressive visual loss and was unable to follow a bright red object at 7 years . ophthalmological examination revealed dystrophic appearance of retina , attenuation of retinal vessels with a pale disc . serial cranial magnetic resonance imaging ( mri ) showed diffuse white matter hyperintensity in t2-weighted but not in flair at 5 years and subsequently , cerebral and cerebellar atrophy at 7 years . electroretinogram ( erg ) was absent and visual evoked potential ( vep ) was normal . palmitoyl protein thioesterase ( ppt ) and tripeptidyl peptidase i ( tpp 1 ) enzyme activities were within normal range . skin and muscle biopsy for inclusion bodies were negative and blood film showed no vacuolated lymphocytes . however , ultra structural examination of lymphocytes showed rare , membrane bound , condensed storage material with fingerprint profiles . cln5 gene mutation was positive . at last follow - up at 11 years , he was wheelchair bound but could walk with support , had severe visual impairment and slow and slurred speech . he was the 6-year - old brother of case 1 and his early developments were normal except for speech . he babbled at 5 months , copied noises at 8 months and only spoke with recognizable word at 2 year 7 months ( his brother spoke at 18 months ) . he would play football ( his eldest brother never ran or jumped ) with other children and his expressive language had never been clear and had difficulty putting words together and had developed stammer . he was slow to learn and at 4 years , he began to have cognitive and motor regression . cranial mri findings were very similar to that demonstrated on his brother 's scan . at last follow - up ( 8 years ) , he had drooling saliva , he was wheel chair bound with deterioration in his speech , cognition , motor skills , and fully dependent for his needs . an 11-year-10-month - old caucasian boy with normal perinatal and neurodevelopmental history was referred to us with 3-year history of progressive visual loss and visual hallucinations . his symptoms started with difficulty in reading , worsening handwriting , and flashes of lights in both eyes ( be ) . he gave us a description and consistently drew figures , which he appeared to be seeing in front of his eyes . the figures had a red border with circles and lines criss - crossing the circles . the va at first presentation was 6/60 in be when he was 10 years . at last follow - up at 13 , va was 6/60 in right eye ( re ) and 3/60 in left eye ( le ) . however , electron microscopy ( em ) of buffy coat showed rare , membrane bound , electron dense , granular material associated with a lipid droplet in the cytoplasm of lymphocytes . ppt activity was low measuring 2.2 nmol / hr / mg protein and normal tpp - i . molecular genetic analysis of exon 1 - 9 of cln1 gene showed that he was heterozygote for p.cys96tyr and p.arg151x mutations . a diagnosis of milder form of incl ( cln1 ) was made . at his last neurology follow - up at 13 years all our cases did not fit well with the typical clinicopathologic descriptions , therefore representing atypical variants of jncl or vlincl due to the late onset at 4 - 7 years and em showed fingerprints , thus , overlapping considerably with juvenile ncl . for case 1 and 2 , similarities to vlincl were age - at - onset , initial symptoms , and clinical progression . differences from classic lincl included slower clinical progression and different inclusion profile ( revealed fingerprint , not typical curvilinear inclusion body ) . in fact , cln2 encodes for ttp-1 enzyme , therefore , normal enzyme level excluded classic lincl ( cln2 ) . furthermore , these two cases had cln5 mutation which usually presents as vlincl and presented with typical phenotype of cln5 . all the cases of ncl caused by mutations in cln1 are associated with granular appearance . to sum up , case 1 and 2 had early juvenile form of ncl ( vlincl ) due to cln5 and case 3 had atypical jncl due to cln1 mutation , reflecting phenotypical - genotypical variation . however , delayed vep , normal erg and fundi were also unusual and were consistent with optic nerve neuropathy . visual loss in cln5 is usually due to retinal dystrophy . ophthalmologic examination early in the course of vlincl ( cln5 ) may reveal macular changes only ; gradually typical signs of pan - retinal degeneration develop : bull 's eye maculopathy , diffuse pigmentary degeneration , arteriolar attenuation , and optic atrophy . on contrast , case 3 ( cln1 ) showed normal erg , delayed vep and persistent normal fundi even at an advanced stage . normal disc may still correspond to an early stage of juvenile form of cln1 but was quite unexpected at this advanced stage ( 4 years duration with va 6/60 ) . patients with cln1 ( case 1 and 2 ) have cerebellar atrophy at the early stage , followed by brain atrophy that is more severe in the cerebellum . in contrast , patients with typical jncl ( cln3 ) have cerebral atrophy at the early stage . atypical jncl or vlincl should be considered in any patients who present with either psychomotor decline or progressive visual loss . phenolic compounds are the most commonly studied of all secondary metabolites because of their significant concentration and their significant roles in plant tissues [ 1 , 2 ] . this highly diverse group of secondary metabolites widely is distributed in vegetable foods ( legumes , cereals , and fruits ) and beverages ( tea , cider , and wine ) , which are important constituents of the human diet . the rapid degradation of flavonoids during the extraction is a major drawback for the accurate quantification of this ubiquitous group of compounds . because of their importance as potent antioxidants molecules , quantification flavonoids in fruits , vegetable , and other crops are important . they become perishable mainly due to the enzymatic oxidation regulated by the enzyme polyphenol oxidase and light [ 5 , 6 ] . polyphenol oxidase is activated when the plant tissues are macerated and the enzymes get mixed with the substrate and the major parts of the native flavonoids are destroyed before quantification . the sustainability of the tea industry mainly relies on developing new tea ( camellia sinensis , l. ) cultivars incorporated with genetic resistance to biotic and abiotic stresses and capable of producing diverse tea products of desired character . the quality of tea product depends on the chemical composition of fresh tea leaves and the method of manufacture . the chemical composition of tea leaves is determined by the inherent genetic attributes of the accession and geoclimatic factors . currently about 600 germplasm accessions are being maintained in the field gene bank of the tea research institute of sri lanka ( tri ) and these accessions are not adequately evaluated for biochemical , agronomic , and molecular traits [ 11 , 12 ] . most of the earlier attempts concentrated on single metabolite variation of tea cultivars , and work on measurement of a broad range of metabolites is relatively limited . thus , metabolite profiling of representative accessions from the sri lankan tea germplasm by chemical analysis of tea leaves would immensely contribute to the success of a tea breeding programme . the polyphenols , particularly catechins ( flavan-3-ols ) and their oxidation products , which are eventually responsible for the quality of tea , are readily oxidized by polyphenol oxidase enzymes . the collection , handling , and storage of a large number of leaf samples for metabolite profiling thus require a sample preparation procedure that would minimize the oxidation of polyphenols and formation of artifacts . in this backdrop , this research has been carried out with the objective to optimize the sample preparation procedure to minimize the chemical deterioration that takes place in tea flush due to the inevitable delay in analysis . for this purpose , a comparative study has been conducted between the fresh leaves ( conventional procedures ) and freeze - dried leaves ( new sampling method ) , for quantification of some critical metabolites gallic acid , catechins , and methylxanthines , by employing two cultivars , one ( dt1 ) of which is known to yield high quality black tea and the other ( tri2025 ) poor quality black tea . caffeine , gallic acid , ( )-epicatechin , ( )-epigallocatechin , ( )-epigallocatechin gallate , and ( )-epicatechin gallate were purchased from sigma chemical co. ( st . louis , mo , usa ) . methanol , acetonitrile , acetic acid , and folin - ciocalteu 's phenol reagent were purchased from bdh , uk . standard reference material ( srm 3254 ) for green tea was obtained from the national institute of standards and testing ( nist ) , usa . a millipore milli - q water purification system was used to obtain high purity of water . two cultivars ( dt1 and tri2025 ) were selected for this study from ex situ field gene bank at the tea research institute of sri lanka , talawakelle ( latitude 654n , longitude 8042e ) . the plants were of same age and have been maintained under similar growth conditions recommended by the tea research institute of sri lanka . five grams of fresh two leaves and a bud was placed in 70% aqueous methanol ( 40 ml ) at boiling temperature for 10 min . the mixture was cooled to room temperature and homogenized for 3 min using a top - drive macerator ( ultra - turrax , usa ) . the homogenate was centrifuged ( 24.15 g for 15 min ) and the supernatant was decanted into a 50 ml volumetric flask . the residue was reextracted following the above extraction procedure with 70% aqueous methanol ( 10 ml ) at boiling temperature . the volume of the pooled extract was made up to 50 ml with 70% aqueous methanol . an aliquot ( 1.0 ml ) of the above methanol extract was transferred into a 200 ml volumetric flask and topped up with milli - q water . approximately 10 g of fresh two leaves and an active bud from each cultivar was harvested and brought to the laboratory in ice box at 4c . the plant material was immediately deep frozen at 80c for 6 h and then freeze - dried for 24 h ( labconco - freezone , usa ) . the freeze - dried flush was ground to a fine powder ( passed through 500 m mesh ) in a laboratory mill , sealed in triple laminated aluminum foil packages , and stored until use at room temperature . 0.2000 0.001 g freeze - dried tea sample was weighed into a centrifugation tube and extracted with 5.0 ml of 70% methanol / water extraction mixture , preheated to 70c for half an hour . heating of the test portions in the water bath was continued for 10 min with mixing on the vortex mixer at the beginning , after 5 min and at the end of 10 min . then the extraction tube was allowed to cool to room temperature and placed in a centrifuge at 1095 g for 10 min . the extraction steps were repeated on the residue , the extracts were combined , and the volume was made up to 10 ml with cold methanol / water extraction mixture and mixed . 1.0 ml of the sample extract was topped up to 5 ml with freshly prepared stabilizing solution containing 500 g ml of edta and 500 g ml ascorbic acid in 10% volume fraction of acetonitrile . the dry matter content was determined by loss in mass at 103c determined on a portion of the test sample in accordance with iso1573 method for both fresh flush leaves and freeze - dried flush . a series of gallic acid standard solutions was prepared with concentrations ranging from 10 to 50 g ml . the tpp in the standards and the extracts were determined in quadruplicate using folin - ciocalteu 's phenol reagent according to iso 14502 - 1 method using a spectrophotometer ( cary 50bio , varian , usa ) set at 765 nm . caffeine , ec , ecg , egc , egcg , gallic acid , and theobromine were quantified in duplicate in two trials of samples using iso 14502 - 2 method by hplc ( agilent 1260 hplc infinity system ) on a phenyl - bonded column using elution with uv detection at 278 nm . the hplc instrument consisted of agilent 1260 infinity hplc system with solvent delivery system , temperature - controlled column oven , autosampler , degasser unit , and a photodiode array detector and open lab / chemstation software system . luna-5 m phenyl - hexyl-4.6 250 mm column ( phenomenex inc . , usa ) with a guard column made of the same material ( security - guard , phenomenex inc . , a was 9% acetonitrile containing 2% acetic acid ( bdh hipersolv - hplc grade ) . solvent gradient was reset to 100% mobile phase a and allowed to equilibrate for 10 mins before the next injection . the elution was reset to 100% mobile phase a and allowed to equilibrate for 10 min before next injection . flow rate was set to 1.00 ml / min and column oven temperature was kept at 35 0.5c . once the flow rate of the mobile phase and temperature were stable , the column was conditioned with a blank gradient run . after each batch of analysis , the chromatographic system and column were thoroughly flushed with 50% aqueous acetonitrile and column sealing plugs were replaced if disconnected for storage . the individual catechins ( ec , ecg , egc , and egcg ) and gallic acid were identified by comparing retention times from sample chromatograms with those recorded for a variety of standards obtained under the same chromatographic conditions . the use of diode array detection allowed the uv profile of the catechin peaks to be scrutinized and peak purity to be assessed , which was particularly useful for the determination of the low levels of catechins in tea cultivars . alternatively , caffeine was used as a standard in conjunction with individual catechins considering the relative response factors ( rrfs ) established by an iso international interlaboratory test . data was collected using the data collection / integration system for all peaks in the extracts . standard reference material ( srm 3254 ) for green tea certified by nist , usa , was used for the validation of the analytical methodology . significant differences ( p < 0.01 ) were tested using two tailed paired t - test at 95% confidence level . least significant difference ( lsd ) was used as the mean separation technique . all these analyses were carried out with the statistical analysis software package ( sas version . 9.1 for windows , sas institute inc . one of the most important fractions in tea flush is the proteins , because they include the enzymes which are required for the metabolism in the tea plant and for black tea manufacture . polyphenol oxidase is an important enzyme available in tea , which is activated by light and is responsible for the oxidation of flavonols in the presence of atmospheric oxygen . in the conventional sample preparation methodology there is an inevitable delay in the analysis during which the tea flush is subjected to mechanical damage . this results in the formation of characteristic compounds of black tea [ 19 , 20 ] . though it is a positive reaction initiated in the rolling process of tea manufacturing , it hinders accurate and precise measurement of metabolites in the tea flush . further , the content of catechins is dependent upon other factors including particularly light since some of the important enzymes of the flavonoid biosynthetic pathway are activated by light . since the analysis of the batch of cultivars sampled has to be done on the same day , it was not possible to handle a large number of samples at a time . in contrast , the new method suppressed chemical deterioration starting from the point of harvest until usage . as described in figure 1 , the samples harvested from the field were immediately collected into and ice box at 4c whereby the chemical deterioration of the metabolites was minimized . prefreezing at 80c for 6 h to two weeks is a convenient first step in sample preparation prior to freeze - drying . freeze - drying is a preservation technique by which moisture is removed and the shelf - life is increased approximately up to one year . the dry matter content of the freeze - dried samples was eight times less than that of the fresh flush . therefore , substantially less amount of sample was required for analysis in the new method ( table 1 ) and convenient handling of many samples was possible at a time . also , freeze - dried leaf samples can be stored for a long time when packed in triple laminated aluminum sheets and it facilitates sampling at once and stored for metabolite analysis . thus , environmental influence on metabolite availability could be minimized specially when handling large number of samples . maximum recovery of the metabolites required a minimum of 70% methanol in the extractant which was sufficient to inactivate polyphenol oxidase . multiple extraction procedures of short durations ( 10 min ) at moderate temperature ( 70c ) in the new method were more favourable than those involving boiling temperature and long extraction times in a macerator as such conditions would lead to decomposition of catechins . efficient separation of caffeine , ( )-epicatechin ( ec ) , ( )-epigallocatechin ( egc ) , ( )-epigallocatechin gallate ( egcg ) , ( )-epicatechin gallate ( ecg ) , gallic acid , and theobromine in the tea extracts ( figure 2 ) was achieved in less than 25 min of a chromatographic run by the gradient elution with acetonitrile , water , and acetic acid . although the sensitivity of the analysis can be increased by threefold by using a shorter detection wavelength , the chance of interference by other components present in the tea extract is high . the folin - ciocalteu 's phenol reagent used for tpp analysis contains phosphotungstic acid as oxidants , which on reduction yield a blue colour with a broad maximum absorption at 765 nm . as this reagent reacts with a wide range of polyphenol compounds , it is often used to estimate the total polyphenol content in plant material rich in polyphenols , although the response can vary with the individual components depending on their chemical structures . however , the folin - ciocalteu 's reagent could simultaneously oxidize several nonphenolic organic compounds as well as some inorganic substances to give elevated apparent phenolic content . as it is based on the reduction properties of sample components the mean values of tpp , and the amounts of four catechins ( ec , ecg , egc , and egcg ) , caffeine , theobromine , and gallic acid recorded for the two cultivars , dt1 and tri2025 , corresponding to the two sampling procedures ( fresh leaves and freeze - dried leaves ) , are given in table 2 . the concentration of each metabolite varied with the cultivar . for both cultivars , dt1 and tri2025 , the contents of all the metabolites ( tpp , ec , ecg , egc , egcg , caffeine , theobromine , and gallic acid ) determined by employing new method were found to be higher than in the conventional method ; the difference was statistically significant ( p < 0.05 ) for each of the metabolites except for theobromine in cultivar dt1 . national institute of standards and testing ( nist ) certified srm 3254 quantified via these two methods confirms that the new method is accurate and precise for the determination of the major metabolites in tea . the freeze - dried sampling procedure recorded significantly higher amount of metabolites with small leaf quantity and facilitates storage of samples without chemical deterioration starting from the point of harvest until usage . hence , the method is more suitable for metabolite profiling of tea germplasm using high throughput analysis where large number of samples need to be handled .

the diagnosis of juvenile neuronal ceroid lipofuscinosis ( jncl ) is usually based on age of onset , initial clinical symptoms , clinical progression , and pathologic findings . our cases manifested atypical clinical symptomatology and/or pathologic findings and therefore , represent variant forms of jncl . case 1 and 2 presented with slow developmental regression from the age of 4 years and became blind and wheelchair bound at around 8 years . pathologic finding of lymphocytes showed fingerprint inclusion which was consistent with jncl . mutational analysis was positive for cln5 which usually presents as variant late infantile ncl ( lincl ) and more common in finnish population . case 3 presented with progressive visual loss from the age of 8 years . clinical symptomatology and age of onset were similar to that of jncl but was found to have low palmitoyl protein thioesterase , granular inclusion body , and cln1 mutation , thus representing milder form of incl . these three cases demonstrated phenotypic - genotypic variations . pertinent issues relating diagnostic difficulties , ophthalmologic , neuroradiological , and laboratory aspects are discussed . chemical analysis of the sri lankan tea ( camellia sinensis , l. ) germplasm would immensely contribute to the success of the tea breeding programme . however , the polyphenols , particularly catechins ( flavan-3-ols ) , are readily prone to oxidation in the conventional method of sample preparation . therefore , optimization of the present sample preparation methodology for the profiling of metabolites is much important . two sample preparation methodologies were compared , fresh leaves ( as in the conventional procedures ) and freeze - dried leaves ( a new procedure ) , for quantification of major metabolites by employing two cultivars , one is known to be high quality black tea and the other low quality black tea . the amounts of major metabolites such as catechins , caffeine , gallic acid , and theobromine , recorded in the new sampling procedure via freeze - dried leaves , were significantly higher than those recorded in the conventional sample preparation procedure . additionally new method required less amount of leaf sample for analysis of major metabolites and facilitates storage of samples until analysis . the freeze - dried method would be useful for high throughput analysis of large number of samples in shorter period without chemical deterioration starting from the point of harvest until usage . hence , this method is more suitable for metabolite profiling of tea as well as other phenol rich plants .

with the aggravation of environmental pollution , lung cancer is almost the most malignant tumor in the world with its high morbidity and mortality.1 the most common histologic subtype is non - small cell lung cancer ( nsclc ) , which accounts for 80% of all lung cancers.2 although much progress has been made in the treatment of lung cancer , early diagnosis is difficult and the majority of patients has progressed to an advanced stage when diagnosed . the median survival rate for these patients is only 811 months.3 in 2004 , a landmark discovery had been made in that somatic mutations in the epidermal growth factor receptor ( egfr ) were associated with sensitivity to egfr tyrosine kinase ( tk ) inhibitors ( tkis ) ( egfr - tki).4,5 in subsequent large - scale randomized clinical trials , the relationship between egfr mutation status and efficacy of the egfr - tki drug was clearly explained.68 based on these findings , egfr mutation status in the tk domain can determine the treatment of advanced nsclc . mutations associated with enhanced sensitivity to egfr - tkis are found in exons 1821 of the tk domain of egfr ; in particular , del e746-a750 in exon 19 and the l858r point mutation in exon 21 account for nearly 90% of all the mutations in egfr in lung cancer.7,9,10 nowadays , the detection of egfr mutation status in nsclc patients has become an expert consensus.11 different methodologies have been developed for molecular testing , such as the amplified refractory mutation system ( arms ) , high - resolution melting , dna direct sequencing , and next - generation sequencing ( ngs ) . dna direct sequencing is considered the gold standard for the assessment of egfr mutation status in nsclc ; however , it is time consuming and laborious . the arms method is widely used in the clinical testing ; however , the commercial assay kit for egfr is very expensive , and the experiment needs to be done under good experimental conditions with sophisticated real - time polymerase chain reaction ( pcr ) instruments.12 immunohistochemistry ( ihc ) is a well - established method that is widely applied in conventional pathological diagnosis . egfr mutation - specific rabbit monoclonal antibodies against e746-a750 deletion and l858r ( cell signaling technology , inc . , danvers , ma , usa ) have been applied in ihc application . this provides a simple and rapid screening method for assessing egfr mutation status.1315 a nanofluorescent material , fluorescent semiconductor nanocrystal quantum dots ( qds ) , have been widely used in labeling some molecules , such as streptavidin and antibodies , through carbodiimide chemistry , optionally using edac ( 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ) . qd ( 605 nm)-labeled streptavidin emits bright red fluorescence with 605 nm as the maximum emission wavelength while being stimulated by an excitation light source < 580 nm , which is different from green background autofluorescence . those labeled materials can be successfully applied to biological imaging and ihc detection of gene mutations , such as with human epidermal growth factor receptor 2 ( her 2 ) amplification in breast cancer.16,17 moreover , multiple markers can be visualized on one cell for in vitro multiplexed imaging ; for example , clinically significant tumor biomarkers including her2 , egfr , progesterone receptor , estrogen receptor , and mammalian target of rapamycin can be detected quantitatively and simultaneously in breast cancer cells using multicolor qds.18 qd - based immunofluorescence histochemistry ( qds - ihc ) is an established method ; it has been validated in many published papers.17,19,20 the staining signal detected by qds - ihc is much stronger with a lower background when compared with ihc.20 based on the application of egfr mutation - specific antibodies , this study was designed to develop a qd - based immunofluorescent approach for egfr mutation detection , which is a simple , quick , and highly sensitive molecular method for diagnosing egfr mutation status in nsclc samples . fifty - five cases of formalin - fixed , paraffin - embedded ( ffpe ) nsclc specimens and ten cases of pleural effusion cell blocks from patients with lung adenocarcinoma were collected , which were provided by the central hospital of enshi autonomous prefecture and the hubei cancer hospital from january 2013august 2014 . the cohort consisted of 13 squamous cell carcinomas , 50 adenocarcinomas , and two adenosquamous carcinomas . for each case , the hematoxylin and eosin sections were reviewed by at least two pathologists ( ygq and hlc ) . this study was approved by the institutional ethics committee of the central hospital of enshi autonomous prefecture . nsclc tissue sections ( 4 m thick ) were deparaffinized in xylene and rehydrated in a graded ethanol series . qds - ihc was performed according to the manufacturer s instructions ( wuhan jiayuan quantum dots co. , ltd . , antigen retrieval was performed in ethylene diamine tetraacetic acid ( edta ) ( 10 mm ; ph 9.0 ) at 100c for 3 minutes , followed by cooling at room temperature for 30 minutes . for antibody bindings , sections were first incubated in 2% bovine serum albumin ( bsa ) buffer ( sigma - aldrich co. , st louis , mo , usa ) at 37c for 30 minutes , and then three primary antibodies ( total egfr monoclonal antibody [ d38b1 ] , egfr del e746-a750 mutation - specific monoclonal antibody [ 6b6 ] , and l858r mutation - specific monoclonal antibody [ 43b2 ] ; cell signaling technology , inc . ) were diluted separately at 1:100 and manually applied to the sections . after that , the slides were then washed three times with tris - buffered saline ( tbs ) with tween ( tbs - t ) ( 0.5% tween , 0.1 m tris - base , 0.9% nacl , and ph 7.6 ) for 5 minutes each time , and incubated in biotinylated goat antirabbit immunoglobulin g ( 1:300 dilution ; jackson immunoresearch inc . , finally , qd ( 605 nm)-labeled streptavidin ( 1:400 dilution in 2% bsa ; wuhan jiayuan quantum dots co. , ltd . ) was added to the sections and incubated at 37c for 30 minutes . after rinsing for three times , sections were sealed with 90% glycerine ( sigma - aldrich co. ) . during the process , negative control samples were performed in parallel , but the primary antibody was replaced with tbs buffer . after washing the sections with tbs , diluted primary antibodies ( 1:100 ) were applied to cover the specimen . after three washes in tbs for 3 minutes each , the slides were incubated for 30 minutes at room temperature with labeled polymer - horseradish peroxidase antirabbit secondary antibody ( envision kit ; dako denmark a / s , glostrup , denmark ) . the qd staining signals were detected using olympus bx51 fluorescence microscopy equipped with an olympus micro dp72 camera ( olympus corporation , tokyo , japan ) . the egfr - positive signal detected by qds - ihc was red , target - specific , bright , and photostable . the egfr - positive signal detected by traditional ihc was brown yellow or brown . immunostaining was evaluated by two different pathologists ( ygq and xdz ) using criteria based on published cutoffs . the intensity of cytoplasmic and/or membrane staining , as well as the percentage of positive cells , was recorded . staining intensity was scored from 0 to 3 + , as follows:21,22 0 if tumor cells had a complete absence of staining or faint staining intensity in < 10% ; 1 + if 10% of the tumor cells had faint staining ; 2 + if the tumor cells had moderate staining ; and 3 + if tumor cells had strong staining ( figure 1 ) . accordingly , we classified scores of 0 and 1 + as negative and scores of 2 + and 3 + as positive . to assess the sensitivity and specificity of qds - ihc , we compared these results with those of real - time quantitative pcr . extraction of genomic dna from ffpe nsclc tissue sections was performed using cobas sample preparation kits ( hoffman - la roche ltd . , the dna quality and purity were assessed using varioskan flash ( thermo fisher scientific , waltham , ma , usa).23 arms is a highly sensitive method ; it is a real - time pcr - based test . the amoydx egfr mutation test kit ( amoy diagnostics co. , ltd . , xiamen , people s republic of china ) has been widely used in the clinical laboratory . the assay was carried out according to the manufacturer s protocol for the kit with the lightcycler 480 ii real - time pcr system ( hoffman - la roche ltd . ) . upon completion , the results were analyzed according to the criteria defined by the manufacturer s instructions . statistical analysis was performed using the statistical software spss version 17.0 ( ibm corporation , armonk , ny , usa ) . cohen s kappa was used to determine intraobserver agreement and the agreement between qds - ihc , ihc , and arms . a kappa value between 0.81 and 1.0 was defined as a nearly perfect agreement , between 0.41 and 0.80 as a moderate agreement , between 0.21 and 0.40 as a fair agreement , and between 0.00 and 0.20 as a slight agreement . all tests were two - sided , and a p - value of < 0.05 was considered statistically significant . fifty - five cases of formalin - fixed , paraffin - embedded ( ffpe ) nsclc specimens and ten cases of pleural effusion cell blocks from patients with lung adenocarcinoma were collected , which were provided by the central hospital of enshi autonomous prefecture and the hubei cancer hospital from january 2013august 2014 . the cohort consisted of 13 squamous cell carcinomas , 50 adenocarcinomas , and two adenosquamous carcinomas . for each case , the hematoxylin and eosin sections were reviewed by at least two pathologists ( ygq and hlc ) . this study was approved by the institutional ethics committee of the central hospital of enshi autonomous prefecture . nsclc tissue sections ( 4 m thick ) were deparaffinized in xylene and rehydrated in a graded ethanol series . qds - ihc was performed according to the manufacturer s instructions ( wuhan jiayuan quantum dots co. , ltd . , antigen retrieval was performed in ethylene diamine tetraacetic acid ( edta ) ( 10 mm ; ph 9.0 ) at 100c for 3 minutes , followed by cooling at room temperature for 30 minutes . for antibody bindings , sections were first incubated in 2% bovine serum albumin ( bsa ) buffer ( sigma - aldrich co. , st louis , mo , usa ) at 37c for 30 minutes , and then three primary antibodies ( total egfr monoclonal antibody [ d38b1 ] , egfr del e746-a750 mutation - specific monoclonal antibody [ 6b6 ] , and l858r mutation - specific monoclonal antibody [ 43b2 ] ; cell signaling technology , inc . ) were diluted separately at 1:100 and manually applied to the sections . specimens were incubated at 4c overnight with those three primary antibodies , respectively . after that , the slides were then washed three times with tris - buffered saline ( tbs ) with tween ( tbs - t ) ( 0.5% tween , 0.1 m tris - base , 0.9% nacl , and ph 7.6 ) for 5 minutes each time , and incubated in biotinylated goat antirabbit immunoglobulin g ( 1:300 dilution ; jackson immunoresearch inc . , finally , qd ( 605 nm)-labeled streptavidin ( 1:400 dilution in 2% bsa ; wuhan jiayuan quantum dots co. , ltd . ) was added to the sections and incubated at 37c for 30 minutes . after rinsing for three times , sections were sealed with 90% glycerine ( sigma - aldrich co. ) . during the process , negative control samples were performed in parallel , but the primary antibody was replaced with tbs buffer . resected tumor specimens were stained simultaneously using these three antibodies according to the manufacturer s instructions . after washing the sections with tbs , diluted primary antibodies ( 1:100 ) were applied to cover the specimen . after three washes in tbs for 3 minutes each , the slides were incubated for 30 minutes at room temperature with labeled polymer - horseradish peroxidase antirabbit secondary antibody ( envision kit ; dako denmark a / s , glostrup , denmark ) . following three washes in tbs the qd staining signals were detected using olympus bx51 fluorescence microscopy equipped with an olympus micro dp72 camera ( olympus corporation , tokyo , japan ) . the egfr - positive signal detected by qds - ihc was red , target - specific , bright , and photostable . the egfr - positive signal detected by traditional ihc was brown yellow or brown . immunostaining was evaluated by two different pathologists ( ygq and xdz ) using criteria based on published cutoffs . the intensity of cytoplasmic and/or membrane staining , as well as the percentage of positive cells , was recorded . staining intensity was scored from 0 to 3 + , as follows:21,22 0 if tumor cells had a complete absence of staining or faint staining intensity in < 10% ; 1 + if 10% of the tumor cells had faint staining ; 2 + if the tumor cells had moderate staining ; and 3 + if tumor cells had strong staining ( figure 1 ) . accordingly , we classified scores of 0 and 1 + as negative and scores of 2 + and 3 + as positive . to assess the sensitivity and specificity of qds - ihc , we compared these results with those of real - time quantitative pcr . extraction of genomic dna from ffpe nsclc tissue sections was performed using cobas sample preparation kits ( hoffman - la roche ltd . , the dna quality and purity were assessed using varioskan flash ( thermo fisher scientific , waltham , ma , usa).23 arms is a highly sensitive method ; it is a real - time pcr - based test . the amoydx egfr mutation test kit ( amoy diagnostics co. , ltd . , xiamen , people s republic of china ) has been widely used in the clinical laboratory . the assay was carried out according to the manufacturer s protocol for the kit with the lightcycler 480 ii real - time pcr system ( hoffman - la roche ltd . ) . upon completion , the results were analyzed according to the criteria defined by the manufacturer s instructions . statistical analysis was performed using the statistical software spss version 17.0 ( ibm corporation , armonk , ny , usa ) . cohen s kappa was used to determine intraobserver agreement and the agreement between qds - ihc , ihc , and arms . a kappa value between 0.81 and 1.0 was defined as a nearly perfect agreement , between 0.41 and 0.80 as a moderate agreement , between 0.21 and 0.40 as a fair agreement , and between 0.00 and 0.20 as a slight agreement . all tests were two - sided , and a p - value of < 0.05 was considered statistically significant . tissue sections from small biopsies , pleural effusion cell blocks , and surgery were successfully stained by egfr mutation - specific antibodies , and these antibodies showed distinct immunoreactivity ( red signals ) for the tumor cells , as presented in figure 1 . a positive signal of the total egfr protein detected by qds - ihc and ihc was moderate to strong in all 65 samples , which was regarded as the positive control ( figure 2 ) . the positive rates for the egfr mutation detected by qds - ihc and ihc were 40.0% ( 26/65 ) and 36.9% ( 24/65 ) , respectively . in the qds - ihc method , 12 ( 46.2% ) patients showed e746-a750-specific staining and 14 ( 53.8% ) patients were l858r mutant - specific ( table 1 ) . figure 2 shows the representative images of the same surgical case of cancer , which carried the l858r mutation and total egfr . the cancer cells were strongly stained by the total egfr antibody ( figures 2a and b ) ; moreover , the cancer cells were positively stained for the anti - l858r antibody ( figures 2c and d ) and negative for the del e746-a750 deletion . simultaneously , egfr mutations were also detected by qds - ihc and ihc using mutation - specific antibodies at the pleural effusion cell blocks ( figures 3a d ) and small biopsies ( figures 4a d ) . egfr mutations detected by qds - ihc and ihc exhibited significant difference between squamous cell carcinoma and adenocarcinoma ( p<0.05 ) . in all the 65 nsclc specimens , the mutation rates for egfr detected by adx - arms was 46.15% ( 30/65 ) , 52.00% ( 26/50 ) for adenocarcinoma , 15.38% ( 2/13 ) for squamous cell carcinoma , and 100% ( 2/2 ) for adenosquamous carcinoma . a significant difference in egfr mutations was observed between adenocarcinoma and squamous cell carcinoma ( p=0.018 ) . adx - arms could detect eleven cases of exon 19 deletion , 14 cases of exon 21 l858r mutations , two cases of exon 20 point mutations ( s768i ) , and one case of exon 20 insertion . both exon changes such as exon 19 deletion and exon 20 t790 m mutations , or exon 18 g719x and exon 20 s768i mutations could be detected simultaneously in the same case ( table 1 ) . thirty - five wild - type egfrs were also noted , and there were two invalid results , mainly due to the fact that there was little tissue available ; as such , the concentration of extracted dna was very low ( 2.54 ng/l and 2.04 ng/l ) . we then compared the mutation status between adx - arms and immunostaining - based egfr . of the 26 patients with positive qds - ihc staining , all of the egfr mutations ( exon 19 deletion and exon 21 l858r mutation ) were detected by adx - arms ; however , adx - arms could detect four cases of exon 18 or exon 20 changes . if the adx - arms results were true , the sensitivity of qds - ihc in detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; the specific for both antibodies was 100.0% ( 26/26 ) . two cases with the exon 21 l858r point mutation , as identified by qds - ihc , were negatively stained by ihc using the egfr mutation - specific antibody . ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) . when detecting egfr mutations , qds - ihc and adx - arms demonstrated perfect consistency ( = 0.882 ; p<0.01 ) . excellent agreement was observed between ihc and adx - arms when detecting egfr mutations ( = 0.826 ; p<0.01 ) . tissue sections from small biopsies , pleural effusion cell blocks , and surgery were successfully stained by egfr mutation - specific antibodies , and these antibodies showed distinct immunoreactivity ( red signals ) for the tumor cells , as presented in figure 1 . a positive signal of the total egfr protein detected by qds - ihc and ihc was moderate to strong in all 65 samples , which was regarded as the positive control ( figure 2 ) . the positive rates for the egfr mutation detected by qds - ihc and ihc were 40.0% ( 26/65 ) and 36.9% ( 24/65 ) , respectively . in the qds - ihc method , 12 ( 46.2% ) patients showed e746-a750-specific staining and 14 ( 53.8% ) patients were l858r mutant - specific ( table 1 ) . figure 2 shows the representative images of the same surgical case of cancer , which carried the l858r mutation and total egfr . the cancer cells were strongly stained by the total egfr antibody ( figures 2a and b ) ; moreover , the cancer cells were positively stained for the anti - l858r antibody ( figures 2c and d ) and negative for the del e746-a750 deletion . simultaneously , egfr mutations were also detected by qds - ihc and ihc using mutation - specific antibodies at the pleural effusion cell blocks ( figures 3a d ) and small biopsies ( figures 4a d ) . egfr mutations detected by qds - ihc and ihc exhibited significant difference between squamous cell carcinoma and adenocarcinoma ( p<0.05 ) . in all the 65 nsclc specimens , the mutation rates for egfr detected by adx - arms was 46.15% ( 30/65 ) , 52.00% ( 26/50 ) for adenocarcinoma , 15.38% ( 2/13 ) for squamous cell carcinoma , and 100% ( 2/2 ) for adenosquamous carcinoma . a significant difference in egfr mutations was observed between adenocarcinoma and squamous cell carcinoma ( p=0.018 ) . adx - arms could detect eleven cases of exon 19 deletion , 14 cases of exon 21 l858r mutations , two cases of exon 20 point mutations ( s768i ) , and one case of exon 20 insertion . both exon changes such as exon 19 deletion and exon 20 t790 m mutations , or exon 18 g719x and exon 20 s768i mutations could be detected simultaneously in the same case ( table 1 ) . thirty - five wild - type egfrs were also noted , and there were two invalid results , mainly due to the fact that there was little tissue available ; as such , the concentration of extracted dna was very low ( 2.54 ng/l and 2.04 ng/l ) . we then compared the mutation status between adx - arms and immunostaining - based egfr . of the 26 patients with positive qds - ihc staining , all of the egfr mutations ( exon 19 deletion and exon 21 l858r mutation ) were detected by adx - arms ; however , adx - arms could detect four cases of exon 18 or exon 20 changes . if the adx - arms results were true , the sensitivity of qds - ihc in detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; the specific for both antibodies was 100.0% ( 26/26 ) . two cases with the exon 21 l858r point mutation , as identified by qds - ihc , were negatively stained by ihc using the egfr mutation - specific antibody . ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) . when detecting egfr mutations , qds - ihc and adx - arms demonstrated perfect consistency ( = 0.882 ; p<0.01 ) . excellent agreement was observed between ihc and adx - arms when detecting egfr mutations ( = 0.826 ; p<0.01 ) . the superior optical and electronic properties of qds over conventional organic dyes , such as high brightness , high photostability , continuous absorption , narrow emission bandwidth , and the ability to simultaneously excite multiple fluorescent colors , make them attractive labels for the development of qds - ihc imaging for multiplexing cancer biomarker detection on ffpe tissues.24,25 in our study , we confirmed that qds - ihc is a simple and standardized method for detecting egfr mutations , and it has high sensitivity and specificity when compared with real - time pcr . in addition , the development of specific antibodies against egfr mutation proteins might be useful for the diagnosis and treatment of lung cancer . in general , patients with advanced nsclc ( stage iiib ) do not benefit from surgery alone and are best managed by initial chemotherapy , chemotherapy plus radiation therapy , or radiation therapy alone . egfr mutation status plays a critical role in the therapeutic decision making for these patients . nowadays , egfr - tkis have been recommended as a first - line therapy in nsclc patients with activating mutations of egfr , including gefitinib , erlotinib , afatinib , and so on . several clinical studies have recently shown that egfr - tkis are superior to chemotherapy in nsclc patients with an egfr - activating mutation.26,27 therefore , it is highly important to evaluate the egfr mutation status in advanced nsclc patients , especially before any clinical therapy decision is undertaken . exon 19 del e746-a750 and exon 21 l858r point mutations represent the majority of egfr mutations.27,28 analysis of egfr mutations has become an important tool for targeted therapy in lung cancer , and recently , many efforts have been made to find a more specific and sensitive method ( including arms and ngs ) to detect them.29,30 when compared with conventional dna direct sequencing , targeted ngs provides a more accurate and clinically useful molecular classification method for lung adenocarcinoma.30 however , these techniques are relatively expensive for routine use in clinical laboratories , and they depend on the quality of the samples . the goal of our study was to evaluate the accuracy and sensitivity of qds - ihc in detecting egfr mutations in nsclc when compared with traditional ihc and arms . our results showed that egfr mutations in 40.0% ( 26/65 ) of nsclc patients were detected by qds - ihc , 12 ( 46.2% ) cases showed e746-a750-specific staining , and 14 ( 53.8% ) patients were l858r mutant - specific . we observed nearly perfect consistency between the positive immunostaining results of qds - ihc and adx - arms when detecting egfr mutations status . the sensitivity of qds - ihc when detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; both demonstrated antibody specificity of 100.0% ( 26/26 ) . however , ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) . despite the small number of cases in our study , we also identified that qds - ihc combined with egfr mutation - specific antibodies to detect an egfr mutation has a high specificity and sensitivity in nsclc patients . furthermore , when compared with adx - arms and traditional ihc , qds - ihc for egfr mutation detection can be performed on very small biopsy specimens and pleural effusion cell blocks . this is especially the case in instances when the number of tumor cells of these precious biopsy samples could not reach dna or rna extraction requirements . according to our knowledge , this was the first report on the detection of egfr mutations using qds - ihc in nsclc patients . our study also showed that these methods could precisely detect egfr exon 19 deletion or exon 21 l858r mutation status ; adx - arms had the best sensitivity , which could also detect exon 18 point mutation and exon 20 insertion . nevertheless , adx - arms was more expensive and required strict conditions for routine use in clinical laboratories , which is difficult to carry out and popularize in a basic hospital setting . lung adenocarcinoma showed higher egfr mutations than squamous cell carcinoma ; this mutation was identified by three methods , and it was found that adenocarcinoma exhibits different biological behaviors and requires a different therapeutic strategy . in the present study , we found that qds exhibit excellent photostability , a broad excitation spectrum , and a long fluorescence lifetime.20,31 qds - ihc could accurately detect egfr mutation protein localization in nsclc . taken together , the qds - ihc technique achieves levels of sensitivity and specificity that are sufficient for detecting egfr mutation signals in ffpe surgery , biopsy , and cell block specimens , while minimizing costs and optimizing therapeutic options . combining this method with adx - arms

backgroundepidermal growth factor receptor ( egfr ) mutation status plays an important role in therapeutic decision making for non - small cell lung cancer ( nsclc ) patients . since egfr mutation - specific antibodies ( e746-a750del and l858r ) have been developed , egfr mutation detection by immunohistochemistry ( ihc ) is a suitable screening test . on this basis , we want to establish a new screening test , quantum dots immunofluorescence histochemistry ( qds - ihc ) , to assess egfr gene mutation in nsclc tissues , and we compared it to traditional ihc and amplification refractory mutation system ( arms).materials and methodsegfr gene mutations were detected by qds - ihc , ihc , and adx - arms in 65 cases of nsclc composed of 55 formalin - fixed , paraffin - embedded specimens and ten pleural effusion cell blocks , including 13 squamous cell carcinomas , two adenosquamous carcinomas , and 50 adenocarcinomas.resultspositive rates of egfr gene mutations detected by qds - ihc , ihc , and adx - arms were 40.0% , 36.9% , and 46.2% , respectively , in 65 cases of nsclc patients . the sensitivity of qds - ihc when detecting egfr mutations , as compared to adx - arms , was 86.7% ( 26/30 ) ; the specificity for both antibodies was 100.0% ( 26/26 ) . ihc sensitivity was 80.0% ( 24/30 ) and the specificity was 92.31% ( 24/26 ) . when detecting egfr mutations , qds - ihc and adx - arms had perfect consistency ( = 0.882 ; p<0.01 ) . excellent agreement was observed between ihc and adx - arms when detecting egfr mutations ( = 0.826 ; p<0.01).conclusionqds - ihc is a simple and standardized method to detect egfr mutations with its high sensitivity and specificity , as compared with real - time polymerase chain reaction . in addition , the development of specific antibodies against egfr mutation proteins might be useful for the diagnosis and treatment of lung cancer . we compared bona - fide human induced pluripotent stem cells ( ipsc ) derived from umbilical cord blood ( cb ) and neonatal keratinocytes ( k ) . as a consequence of both incomplete erasure of tissue - specific methylation and aberrant de novo methylation , cb - ipsc and k - ipsc are distinct in genome - wide dna methylation profiles and differentiation potential . extended passage of some ipsc clones in culture did n't improve their epigenetic resemblance to esc , implying that some human ipsc retain a residual epigenetic memory of their tissue of origin .

to investigate the anti - ulcerogenic properties of the chloroform extract of flemingia strobilifera root in rats . other anti - ulcer related activities of the extract such as the effects on free radicals and antimicrobial activity were also evaluated . chloroform extract of flemingia strobilifera root was found to be safe up to 300 mg / kg body weight when administrated orally in female wistar rats . pretreatment with chloroform extract of flemingia strobilifera root reduced the characteristic lesions in a dose dependent manner ( p<0.001 ) when compared with the control . pretreatment with chloroform extract of flemingia strobilifera root at a dose of 15 and 30 mg / kg body wt . increased the gastric mucosal glutathione level , total protein content significantly ( p<0.001 ) as compared to control group . whereas there is significant ( p<0.05 , p<0.001 ) reduction in gastric mucosal malonaldehyde levels when compared to control . free radical scavenging activity of chloroform extract of flemingia strobilifera root was observed in the concentration range tested , the ic50 value was calculated . antimicrobial activity of the chloroform extract of flemingia strobilifera root exhibited activity against both gram positive and negative bacteria at concentration of 10 mg / ml . the root extract of flemingia strobilifera possess antiulcerogenic properties could justify folklore uses of the plant in peptic ulcer diseases . lead is a soft , pliable , bluish - grey metal resistant to corrosion , that exists in both organic and inorganic forms . this metal does not conduct electricity and it owns antiradiation properties . lead poisoning in children is an important health problem , accounting for 0.6% of the global burden of the disease according to the world health organization . according to the national health and nutrition examination survey data , from 2007 to 2010 , approximately 535,000 children aged 1 to 5 years , meaning 2.6% , presented blood lead level above 5 g / dl . even though lead is everywhere the ways of contamination include ingestion , inhalation , prenatal exposure , and dermal exposure , but the most important and frequent ones are ingestion and inhalation . the half - life of lead is between 30 and 40 days in men , while in children and pregnant women it can be longer . it binds to the sulfhydryl group of proteins leading to toxicity for multiple enzyme systems . the clinical presentation of lead poisoning involves nervous , hematologic , and renal systems impairment , but it can also lead to gastrointestinal disorders ( anorexia , vomiting , constipation , abdominal pain ) , hypertension , and fertility impairment . neurological symptoms include ataxia , stupor , coma , convulsions , hyperirritability , reduced iq , shortened attention span , increased antisocial behavior , reduced educational attainment , and even death . impairment of the hematological system may involve either disruption of heme synthesis or hemolysis , leading to anemia with its specific clinical signs , like weakness and fatigue . the effects of lead on the renal system consist in proximal tubular function impairment leading to aminoaciduria , glycosuria , and hyperphosphaturia , interstitial nephritis in chronic exposure , and also impairment of calcium metabolism by interfering with activation of vitamin d 1,2-dihydroxy cholecalciferol . the diagnosis is established on the blood lead level , higher than 40 g / dl for occupational and 30 g / dl for nonoccupational exposure . primary and secondary preventions should be the first steps in the management of lead poisoning as public health problem . if a patient is found with high blood lead level , the test must be repeated before considering any therapy . chelating agents are recommended only if the level is above 45 g / dl , and the type will be chosen according to the blood level and symptoms . the available agents nowadays include : 2,3 dimercaptosuccinic acid ( dmsa ) , dimercaprol , ethylene diamine tetra - acetic acid ( cana2edta ) , d - penicillamine . in certain cases , the management can also include supportive therapy , like airway protection in acute encephalopathy or antiepileptic drugs in case of seizures . we present a case report of lead poisoning in a 16-year - old girl with the aim of highlighting the difficulty in diagnosing this condition , and the fact that even though occupational exposure is the main cause of lead poisoning in adults by inhalation , it can also be present in children in certain circumstances , as in our case . the informed consent was given by the patient 's father ( legal guardian ) for publication of this case report . we present the case of a 16-year - old girl , admitted to our clinic with severe abdominal pain , loss of appetite , nausea , and vomiting . the anamnesis revealed that the girl comes from a family of potters , and that she also participated in the process of pottery , her father being diagnosed with lead poisoning 2 years before . the patient 's personal history underlined that approximately 1 year ago she presented with severe abdominal pain , being diagnosed with acute appendicitis and she underwent appendectomy , but the pain persisted , thus due to family history of lead poisoning , the suspicion of saturnine colic rose , and she was diagnosed with lead poisoning ( urinary lead : 219 g / l ) , but she received only symptomatic treatment . approximately 3 weeks before admission to our clinic , she was admitted to the regional hospital with another episode of saturnine colic ( blood lead : 113.2 g / dl ) , and chelation therapy with edta ( 4 days before the admission in our clinic ) was initiated , with a dose of 2 tablets daily , one in the morning and one in the evening associated with calcium supplements . the clinical examination performed at the time of admission revealed the following pathological elements : influenced general status , ailing face , jaundice of the sclera , blue pigmentation of the nails , painful abdomen at palpation , and weight : 45 kg . the laboratory test performed upon admission revealed hypochromic anemia ( hemoglobin ( hb ) : 10.9 g / dl , hematocrit ( htc ) : 31.6% , medium cellular volume ( mcv ) : 77.6 fl ) , increased level of liver transaminases ( alanine - aminotransferase ( alat ) : 158.9 u / l , aspartate - aminotransferase ( asat ) : 63 u / l , gamma - glutamyl - transferase ( ggt ) : 128 u / l ) , conjugated hyperbilirubinemia ( direct bilirubin ( dbi ) : 1.432 mg / dl ) , hyponatremia ( na : 132 mmol / l ) , and hypopotassemia ( k : 2.85 mmol / l ) . the systolic arterial pressure was 156 mm hg , and the diastolic was 96 mm hg . the blood lead level was 66.28 g / dl , the urinary one was 419.7 g / l ( normal < 50 g / l ) and the value of delta - aminolevulinic acid was 7.66 mg / l ( normal we also performed abdominal ultrasound which revealed a disappearance of the delimitation between the cortical and medullar parts in both kidneys . we requested consultation from an occupational healthcare specialist , who recommended the continuation of chelation therapy with edta , increasing the dose at 4 tablets / day . we also required a neurological consultation , and the specialist established the diagnosis of behavioral disorders with depressive elements , and recommended psychotherapy . based on all these clinical and laboratory findings , we established the diagnosis of lead poisoning . hydration in order to favor lead elimination , approximately 3 liters per 24 hours initially , and we decreased progressively the quantity once she ceased to vomit , and she was able to consume liquids . we associated diuretics , initially furosemide by vein , but the values of the arterial pressure persisted above the upper limit , therefore we were forced to introduce also an angiotensin - converting enzyme inhibitor , with the remission of arterial hypertension . regarding the liver function , we administered amino acids intravenously , associated with liver protectors by mouth . we also administered vitamins of the b complex in order to improve the neurological impairment . the evolution was slightly favorable , in the first 3 days after the admission , the patient continued to present severe abdominal pain , vomiting , and she also complained of pain in the lumbar area . all the laboratory parameters presented normalization of the values after approximately 10 days of treatment . on the 6th day of admission the blood level was 45.57 g / dl , and the urinary one was 836.4 after 14 days of admission , the patient was discharged without any complaints , and we recommended no further exposure to lead , avoiding the contact and the working in the pottery process . the long - term outcome of this case depends on further exposure to this heavy metal . nevertheless , we intend to repeat the blood lead levels after 12 and 24 months , assessing also the renal ( urea , creatinine , urinary exam ) and hepatic functions ( asat , alat , ggt , bilirubin ) . we present the case of a 16-year - old girl , admitted to our clinic with severe abdominal pain , loss of appetite , nausea , and vomiting . the anamnesis revealed that the girl comes from a family of potters , and that she also participated in the process of pottery , her father being diagnosed with lead poisoning 2 years before . the patient 's personal history underlined that approximately 1 year ago she presented with severe abdominal pain , being diagnosed with acute appendicitis and she underwent appendectomy , but the pain persisted , thus due to family history of lead poisoning , the suspicion of saturnine colic rose , and she was diagnosed with lead poisoning ( urinary lead : 219 g / l ) , but she received only symptomatic treatment . approximately 3 weeks before admission to our clinic , she was admitted to the regional hospital with another episode of saturnine colic ( blood lead : 113.2 g / dl ) , and chelation therapy with edta ( 4 days before the admission in our clinic ) was initiated , with a dose of 2 tablets daily , one in the morning and one in the evening associated with calcium supplements . the clinical examination performed at the time of admission revealed the following pathological elements : influenced general status , ailing face , jaundice of the sclera , blue pigmentation of the nails , painful abdomen at palpation , and weight : 45 kg . the laboratory test performed upon admission revealed hypochromic anemia ( hemoglobin ( hb ) : 10.9 g / dl , hematocrit ( htc ) : 31.6% , medium cellular volume ( mcv ) : 77.6 fl ) , increased level of liver transaminases ( alanine - aminotransferase ( alat ) : 158.9 u / l , aspartate - aminotransferase ( asat ) : 63 u / l , gamma - glutamyl - transferase ( ggt ) : 128 u / l ) , conjugated hyperbilirubinemia ( direct bilirubin ( dbi ) : 1.432 mg / dl ) , hyponatremia ( na : 132 mmol / l ) , and hypopotassemia ( k : 2.85 mmol / l ) . the systolic arterial pressure was 156 mm hg , and the diastolic was 96 mm hg . the blood lead level was 66.28 g / dl , the urinary one was 419.7 g / l ( normal < 50 g / l ) and the value of delta - aminolevulinic acid was 7.66 mg / we also performed abdominal ultrasound which revealed a disappearance of the delimitation between the cortical and medullar parts in both kidneys . we requested consultation from an occupational healthcare specialist , who recommended the continuation of chelation therapy with edta , increasing the dose at 4 tablets / day . we also required a neurological consultation , and the specialist established the diagnosis of behavioral disorders with depressive elements , and recommended psychotherapy . based on all these clinical and laboratory findings , we established the diagnosis of lead poisoning . hydration in order to favor lead elimination , approximately 3 liters per 24 hours initially , and we decreased progressively the quantity once she ceased to vomit , and she was able to consume liquids . we associated diuretics , initially furosemide by vein , but the values of the arterial pressure persisted above the upper limit , therefore we were forced to introduce also an angiotensin - converting enzyme inhibitor , with the remission of arterial hypertension . regarding the liver function , we administered amino acids intravenously , associated with liver protectors by mouth . we also administered vitamins of the b complex in order to improve the neurological impairment . the evolution was slightly favorable , in the first 3 days after the admission , the patient continued to present severe abdominal pain , vomiting , and she also complained of pain in the lumbar area . all the laboratory parameters presented normalization of the values after approximately 10 days of treatment . on the 6th day of admission the blood level was 45.57 g / dl , and the urinary one was 836.4 after 14 days of admission , the patient was discharged without any complaints , and we recommended no further exposure to lead , avoiding the contact and the working in the pottery process . the long - term outcome of this case depends on further exposure to this heavy metal . nevertheless , we intend to repeat the blood lead levels after 12 and 24 months , assessing also the renal ( urea , creatinine , urinary exam ) and hepatic functions ( asat , alat , ggt , bilirubin ) . lead poisoning is a severe condition with potential multiorgan damage and even death if lead is present in large amounts in the blood , representing a major health problem . in children , there is no safe lead level , although according to the world health organization , this problem represents approximately 0.6% of the global burden of disease . the former is more common in children due to their tendency of chewing everything , while the latter is more frequent in occupationally exposed adults . in the case presented here , lead entered the blood by inhalation as occupational risk due to the fact that our teenager patient had helped her parents in the pottery process . other occupations with an increased risk for lead toxicity can be battery manufacturing and recycling plants , demolition , remodeling , and renovation projects , rubber and plastic industries , ammunition and manufacturing , automotive/ radiator repair , lead soldering and welding , painting , plumbing , and so on . there are certain storages of lead in the organism , such as bones , teeth , hair , and nails , where it is bound tightly and it does not seem to be harmful since it is not available to other tissues . however in children , only 70% of the absorbed lead will end up in the bones , in comparison with adults in whom 94% will deposit there , fact that can be responsible for the more expressed clinical effects of this condition in small ages . according to lamas and collaborators , it seems that the deposit of active metals , like cadmium and lead , represents a major risk factor for cardiovascular disease . therefore , chelation therapy with edetate disodium can provide important benefits for those with atherosclerotic cardiovascular disorders . it seems that this metal is also incriminated for reducing the bioavailability of nitric oxide , and therefore promoting oxidative stress and inflammation . a recent study assessed the relationship between blood lead levels and childhood asthma , 2 conditions frequently encountered in small children , both related to environmental factors . the study concluded that even though blood lead levels are not significantly associated with asthma diagnosis , the elevated blood levels of this metal lead to a more severe form of asthma in children , being related to eosinophilia and elevated immunoglobulin e levels . the toxicity of blood lead levels on the nervous system is a major concern for children 's normal development . according to the data of the agency for toxic substances and disease registry ( atsdr ) , blood lead level in children , it seems that a blood lead level of 70 to 80 g / dl or greater represents a serious risk . the neurological sequelae of lead poisoning include reduced iq , shortened attention span , increased antisocial behavior , and reduced educational attainment . it was proved that an average increase of blood lead level of 10 g / dl in children will lead to an iq decline between 1.9 to 3.2 points . other sequelae of chronic lead exposure consist in interstitial nephritis , reduction of sperm concentration , total sperm counts , and total sperm motility . on a study performed on 769 american adolescents , fadrowski found that decreased kidney function is positively correlated to a blood lead level less than 10 g / dl , therefore the us government recommends repeated blood lead level testing at 12 and 24 months . we also intend to repeat the blood lead level in our patient at 12 and 24 months . a study performed in developing countries showed that in many latin american countries , lead - glazed ceramics represent a major source of exposure for those who live there . even though the most common cause of lead poisoning in adults is represented by occupational exposure , in our case we detected elevated blood lead levels in a teenage girl after occupational exposure due to the fact that the patient supported the family pottery business . the pottery - making process involves ceramic glazes and decorative paints , which both contain lead . therefore , these hand - made traditional products can represent a real danger not only for potters , but also for those using these products . the diagnosis establishment of this condition can represent a challenge for every physician , especially for a pediatrician due to the fact that the initial symptoms are nonspecific , such as abdominal pain , anorexia , or irritability , suggesting a gastrointestinal pathology . prevention of lead exposure is the most important step in lead poisoning management due to the fact that the neurocognitive damages induced by lead toxicity are irreversible , and also because in children , this metal is much more easily stored than in adults and it impairs the function of multiple organs , such as kidneys , bones , blood , and the brain . for example , in the united states , in 1988 , control disease center ( cdc ) childhood lead poisoning prevention program was created which provided local governments lead poisoning programs . these programs include public health education , policy development , screening protocols , and case management guidelines . we think that it would be of great importance for the potters to benefit at least from public health education about lead toxicity and screening programs for lead poisoning . the screening for lead poisoning would be an important diagnostic tool for the family members involved in the pottery process , diminishing the rate of unreported cases , and also due to the cluster occurrence of this condition . due to the multiple system involvement therefore , the patient submitted to chelation therapy must be closely monitored , because even the elimination of this heavy metal itself can impair the renal function . even though lead is a toxic metal that is easily stored in the body , its removal being almost impossible , chelation therapy remains the standard treatment of lead poisoning . lead toxicity is a life - threatening condition because of its severe acute and chronic complications . in children , there is no safe blood lead level , prevention methods are , therefore , very important in order to avoid toxic multiorganic effects of this metal . even though the diagnosis represents a challenge in case of children mostly due to its rare incidence in teenagers , a physician must always include this possibility in the differential diagnosis for cases with suggestive symptoms .

purpose : to investigate the anti - ulcerogenic properties of the chloroform extract of flemingia strobilifera root in rats.methods:anti-ulcer effect was evaluated by water immersion induced ulcer in rats . other anti - ulcer related activities of the extract such as the effects on free radicals and antimicrobial activity were also evaluated.results:chloroform extract of flemingia strobilifera root was found to be safe up to 300 mg / kg body weight when administrated orally in female wistar rats . water immersion stress produced characteristic lesions in the glandular portion of the rat stomach . pretreatment with chloroform extract of flemingia strobilifera root reduced the characteristic lesions in a dose dependent manner ( p<0.001 ) when compared with the control . pretreatment with chloroform extract of flemingia strobilifera root at a dose of 15 and 30 mg / kg body wt . increased the gastric mucosal glutathione level , total protein content significantly ( p<0.001 ) as compared to control group . whereas there is significant ( p<0.05 , p<0.001 ) reduction in gastric mucosal malonaldehyde levels when compared to control . free radical scavenging activity of chloroform extract of flemingia strobilifera root was observed in the concentration range tested , the ic50 value was calculated . antimicrobial activity of the chloroform extract of flemingia strobilifera root exhibited activity against both gram positive and negative bacteria at concentration of 10 mg / ml.conclusion : the root extract of flemingia strobilifera possess antiulcerogenic properties could justify folklore uses of the plant in peptic ulcer diseases . abstractbackground : lead is a toxic element of the environment which leads to major complications once it enters the blood stream , affecting multiple organs and systems of the body.methods:we present the case of a 16-year - old girl , diagnosed with lead poisoning after occupational exposure due to the fact that the girl was actively involved in the family 's pottery business.history revealed that the girl participated in the process of pottery , her father was also diagnosed with lead poisoning 2 years before . the patient 's personal history underlined that approximately 1 year ago she presented with severe abdominal pain , being diagnosed with acute appendicitis and she underwent appendectomy , but the pain persisted , thus due to family history of lead poisoning , the suspicion of saturnine colic rose , and she was diagnosed with lead poisoning . the main symptoms and signs were severe abdominal pain , vomiting , and arterial hypertension . the clinical evolution was favorable under symptomatic treatment and chelation therapy.results:lead toxicity is a life - threatening condition because of its severe acute and chronic complications . in children , there is no safe blood lead level , prevention methods are , therefore , very important in order to avoid toxic multiorganic effects of this metal.conclusion:even though the diagnosis of lead poisoning remains difficult in children , it must also be taken into consideration by the clinician facing a child with gastrointestinal or neurological involvement .

atypical haemolytic uraemic syndrome ( ahus ) is an orphan disease , with an incidence of 25 cases per million [ 1 , 2 ] . ahus is characterized by haemolytic microangiopathic anaemia , thrombocytopaenia and acute renal failure ( arf ) , with signs or symptoms of thrombotic microangiopathy ( tma ) in other organs . it is due to uncontrolled activation of the alternative pathway of the complement system , caused by genetic mutation in complement genes ( > 60% of patients ) , anti - fh antibodies ( 56% of patients ) and other mutations not related to the complement system recently described , as dkge gene mutation or methylmalonic aciduria . approximately one - third of patients remain without identified mutation [ 35 ] . clinical manifestation of ahus can be triggered by other complement - amplifying conditions ( infection , pregnancy , organ transplant , malignant hypertension and systemic lupus erythematosus ) that can increase the risk of misdiagnosis . onset of symptoms can be sudden and present as the full triad described above . however , clinical disease manifestation can also progress more slowly with mild anaemia , progressive renal insufficiency and oscillating thrombocytopaenia . the kidney is the most frequent organ involved , although manifestations can occur in the brain ( 1020% of cases ) , heart , lungs and gastrointestinal tract . diagnosis of ahus relies on exclusion ; no evidence of shigatoxin - hus , no criteria for thrombotic thrombocytopaenic purpura ( normal adamts13 activity ) and no medical history indicating other causes of tma ( e.g. malignancy , drug use , etc . ) . plasma exchange ( pe ) has been the standard first - line therapy , with variable efficacy . published studies have shown that up to 40% of patients die or progress to end - stage renal disease ( esrd ) during the first episode . eculizumab , a monoclonal antibody directed against c5 that blocks the cleavage of c5 into c5a and c5b , has demonstrated better efficacy and tolerability in the treatment of ahus . we report a case of ahus with long evolution , subclinical haemolytic signs , progressive rf and neurological impairment resistant to pe , which showed a full reversal of neurological lesions after initiation of eculizumab therapy . . family history : her sister had been diagnosed with membranoproliferative glomerulonephritis ( mpgn ) at age 14 . over the disease course , she had anaemia , increased lactate dehydrogenase ( ldh ) and decline in c3 . epilepsy was diagnosed and well controlled , but she developed neurological impairment with progressive mental retardation . magnetic resonance imaging ( mri ) revealed ischaemic lesions in small vessels and progressive renal failure developed over 11 years . she started peritoneal dialysis in 2010 and suffered further neurological impairment and died due to a respiratory infection in august , 2012 . our 27-year - old patient was diagnosed with arf , anaemia and thrombocytopaenia aged 1 year . she recovered spontaneously without sequalae , and no new episodes of anaemia , thrombocytopaenia , ldh and bilirubin increase nor renal impairment was observed during 14 years . at age 15 , she presented with hyperuricaemia , non - nephrotic proteinuria ( 2 g/24 h ) , arterial hypertension [ blood pressure ( bp ) 160/90 mmhg ] and anaemia [ haemoglobin ( hb ) 11.5 g / dl ] , without thrombocytopaenia or schistocytes in peripheral blood . serum creatinine ( scr ) was 88.4 mol / l , and creatinine clearance ( clcr ) was 96 ml / min . renal biopsy showed focal proliferative glomerulonephritis , affecting 28% of glomeruli , with mesangial matrix and cellularity proliferation , and focal hyaline material deposits , conditioning sclerosis in one of the glomeruli . on immunofluorescence , there were intense igm and weak cq deposits ( figure 1a ) . her scr was stable , and no evidence of tma was found ( normal platelet count , no schistocytes , normal bilirubin ) other than a slight and maintained increase in ldh possibly indicating ongoing haemolysis . she also required angiotensin - converting enzyme inhibitors for proteinuria treatment . when she was 26 , arterial hypertension , proteinuria and anaemia worsened , and serum creatinine increased to 176.8 mol / l , with poor response to antiproteinuric therapy . five months later , she had a hypertensive emergency ( bp 216/120 mmhg ) , with hypertonic hypertensive retinopathy on funduscopy , kidney failure ( scr 366.86 mol / l ) , haemolytic microangiopathic anaemia ( hb 6.7 g / dl , ldh 350 ui / l , haptoglobin < 5 mg / dl and schistocytes ) and severe thrombocytopaenia ( 22 109/l ) . coombs test and autoimmunity were negative , complement component c3 was normal ( 85 mg / dl ) and adamts13 activity was 83% . a misdiagnosis of tma secondary to hypertensive emergency in the context of uncontrolled glomerulopathy was made . a kidney biopsy showed advanced tma , with acute and subacute lesions over chronic vascular involvement ( figure 1b and figure 2 ) . the patient remained hypertensive on dialysis , requiring seven drugs to control bp ( table 1 ) . three months later , she presented with mild neurological symptoms ( decreased intellectual performance , a self - limited episode of loss of consciousness ) and a new episode of tma with c3 consumption ( 68 mg / dl ) . an mri was performed , showing several high - intensity subcortical white matter lesions in the frontal lobes , with the appearance of hypoxic - ischaemic or inflammatory lesions . mild cerebral atrophy was also present ( figure 3a ) . despite a neurological evaluation , no other aetiology than tma genetic investigation showed no mutation in fh , fi and mcp genes , but she is homozygotic for a risk haplotype ( h3 ) . based on the patient 's past history , the patient 's sister bad evolution and the presence of a new episode of tma while on dialysis , with better bp control , we reconsider our previous diagnosis of tma secondary to malignant hypertension to ahus being the primary cause and probably also in the sister . after seven sessions without response and persistent haemolytic signs ( marked thrombocytopaenia , anaemia , increased ldh and schistocytes in peripheral blood sample ) , eculizumab was initiated ( 900 mg / week , during 4 weeks followed by 1200 mg/14 days ) . after the third dose , a repeat mri revealed improvement of the cerebral lesions ( figure 3b ) . after 18 months of eculizumab therapy , the patient is still requiring dialysis , but signs of haemolysis and neurological symptoms are completely absent . anaemia is controlled ( hb 12.5 g / dl ) with a lower esa requirement ( darbepoetin 30 g / weekly ) . bp has improved after 2 months of eculizumab and normal bp is maintained with four antihypertensive drugs . table 1.changes in biochemical parameters and treatmentperiod200111 ( clinical stability)dec 2011apr 2012 ( clinical worsening)may 2012 ( hospital admission)jun 2012 ( start dialysis)aug 2012 ( dialysis , stability)oct 2012 ( dialysis , neurological disorders)jun 2014 ( dialysis , stability)scr ( mol / l)88.4176.8366.86750proteinuria ( g/24 h)1.032.212.71n.d.n.d.n.d.n.d.bp ( mmhg)140/85160/90216/120190/100145/80170/110140/80hb ( g / dl)12 19 0.56.78 0.510.69.512.5platelets ( cells/10 l)256 40231 40224 3022 1521549 20157 20ldh ui / l ( normal range : 125243)265 10300 20377500354363216haptoglobin ( mg / dl)<512579schistocytes ( per hpf)absentabsent2/hpf2/hpfabsentabsentabsenttreatment darbepoetin80 20 g / month80 g/15d80 g/15d150 g / week10 g/15d150 g / week30 g / week aceiramipril 10 mg / bidramipril 10 mg / bid arbsirbesartan 300 mg / dirbesartan 300 mg / d other antihypertensiveamlodipine 5 mg / dhydrochlorotiazide ( hctz ) 25 mg / damlodipine 5 mg / dhctz 25 mg / ddoxazosin 4 mg / bidmethyldopa 500 mg / bidamlodipine 5 mg / dtorasemide 5 mg / ddoxazosin 4 mg / bidmethyldopa 500 mg / bidnebivolol 5 mg / dbarnidipine 10 mg / bidfurosemide 80 mg / dhctz 25 mg / ddoxazosin 4 mg / bidlabetalol 100 mg / bidhydralazine 50 mg / tidbarnidipine 10 mg / bidlabetalol 100 mg / bidhydralazine 50 mg / tidaliskiren 150 mg / dbarnidipine 10 mg / bidfurosemide 40 mg / ddoxazosin 4 mg / bidatenolol 25 mg / bidbarnidipine 10 mg / bidfurosemide 40 mg / ddoxazosin 4 mg / bidatenolol 25 mg / bid pe5 sessions7 sessions eculizumab900 mg / week1200 mg/14dn.d . , not documented ; d , day ; bid , twice a day ; tid , three times a day . focal proliferative glomerulonephritis , with mesangial matrix and cellular proliferation , and focal hyaline material deposits . signs of acute thrombotic microangiopathy with endoluminal collapse , endothelial and myointimal proliferation . fig . 3.evolution of mri before ( a ) and after ( b ) . ( a ) mri before eculizumab therapy . , not documented ; d , day ; bid , twice a day ; tid , three times a day . focal proliferative glomerulonephritis , with mesangial matrix and cellular proliferation , and focal hyaline material deposits . evolution of mri before ( a ) and after ( b ) . ( a ) mri before eculizumab therapy . apart from the efficacy of eculizumab for controlling neurological manifestations of ahus , the present case highlights the difficulties in diagnosing ahus when signs of the disease are mild ( anaemia with subclinical haemolytic signs , gradual impairment of renal function and the absence of thrombocytopaenia ) and progress slowly . usually , ahus has an abrupt onset , with a complete clinical picture that helps the differential diagnosis . the lack of thrombocytopaenia , schistocytes and worsening anaemia until the last episode of kidney failure made diagnosis difficult . slow disease development has been described in 20% of patients , with proteinuria , elevated bp and progressive renal failure . in relapsing / remitting disease our patient was initially diagnosed with focal glomerulosclerosis with igm deposits and non - nephrotic proteinuria ( but she had a previous episode of arf , probably due to tma episode ) . her sister was diagnosed with mpgn . mpgn and ahus may sometimes look similar on a biopsy and partially recovered tma could possibly mimic chronic glomerulonephritis . the association between them has been made as both are diseases related to aberrant complement activation . proteinuria could be explained by endothelial dysfunction caused by tma , but nephrotic syndrome itself can trigger ahus by inducing endothelial injury in patients with genetic mutations [ 13 , 14 ] . moreover , alternative pathway dysregulation can manifest only as low - grade proteinuria and chronic kidney disease [ 13 , 15 ] . vasculitis can trigger ahus by intensive complement activation or by endothelial damage in a patient with a genetic predisposition . in our case , glomerulonephritis was not the initial event ; she had a prior episode of arf . it is possible that partially recovered tma episodes mimic chronic glomerulonephritis on biopsy , as in the present case . the non - classical presentation in both sisters made diagnosis of ahus as a primary disease complex . the biopsy findings were in addition not entirely conclusive for a diagnosis of ahus , as the biopsy showed focal proliferative glomerulonephritis . the importance of risk polymorphisms in the development of ahus is increasingly being recognized , and a lack of identified complement mutations is observed in 2530% of patients [ 3 , 17 ] . the similar evolution in the two sisters , and the good response to eculizumab in our patient , suggests a complement system disturbance , despite the absence of identified mutations . the evolution of the two patients supports the evidence that prognosis for ahus with no identified mutation is as poor as in patients with identified complement factor mutations , in the absence of adequate therapy . the prognosis for patients with ahus before eculizumab was poor , with up to 40% of patients dying or progressing to esrd during the first episode . renal function recovery has been described with eculizumab even after 6 months of dialysis . in our case , kidney function did not improve despite treatment , probably due to the long - term nature of the organ damage causing irreversible damage and the delay in starting eculizumab therapy . in our patient , neurological damage progressed despite cessation of clinical haemolysis and esrd . therefore , the presence of extrarenal symptoms is critical when deciding to maintain anti - c5 therapy , regardless of renal replacement therapy requirement . damage to extrarenal organs can progress in patients without renal function [ 21 , 22 ] . this indicates subclinical activity , showing that an increase in platelet count is not always a reliable marker of recovery , highlighting the need for other biomarkers of disease activity . in the present case , rapid neurological recovery after eculizumab has been described previously , and it is maintained over time [ 20 , 22 , 24 , 25 ] . in conclusion , nephrologists should be aware of the possibility of ahus with a slow , low - grade chronic evolution that can be difficult to diagnose . our case suggests that neurological involvement can be reversed with eculizumab that is becoming the standard of care in ahus therapy , even in patients with ahus on dialysis . severe accidental hypothermia is a condition associated with significant morbidity and mortality . in the years 20092012 the polish national statistics department reported 1836 deaths due to excessive exposure to natural cold . the severe accidental hypothermia center ( clhg centrum leczenia hipotermii glebokiej ) was set up in krakow in 2013 ( darocha , 2015 ) . it is a unit functioning within the structure of the cardiac surgery clinic , established in order to improve the effectiveness of the treatment of patients in the advanced stages of severe hypothermia . the coordination of rescue operations ( over an area of 32,950 square km ) was entrusted to a group of doctors ( 4 coordinators ) , who support the decision - making and organization of the operational process from the time of obtaining the information about the incident until reaching clhg . on december 29 , 2014 at 19:15 , the rescuer on duty at the mountain rescue service ( gopr ) received information about a group of tourists who had lost their way in the top regions of babia gora mount , at the height of about 1620 meters above sea level . air temperature was between 6 and 8c ( windchill factor below 0c ) , the wind was w - wnw with gusts up to over a few dozen km per hour , and the falling snow significantly reduced visibility . the mountain rescue service search party on duty ( two people ) left immediately to conduct the search . later they were joined by others , so that 19 mountain rescue service members eventually took part in the operation . at 19:30 , in accordance with the protocol , the member of the mountain rescue notified the severe accidental hypothermia center ( clhg ) about the search operation . at 20:55 two men were conscious and in stage i of hypothermia , while the third one , sitting in the snow , was confused , agitated , and aggressive towards the rescuers who suspected the ii / iii stage of hypothermia according to the swiss staging system . information about this situation was passed on by phone to the clhg coordinator by the gopr rescuers . the victims were covered with metalized foil , put in thermal sleeping bags and in akia emergency rescue sleds . external warming by means of heating pads was started , and continuous heart rate monitoring using an aed was implemented . at 22:08 the mountain rescue service ( gopr ) rescuers started the difficult transport of the victims downhill , in the direction of the meeting place agreed upon with the teams of emergency medical services who were to take further care of the victims . on the basis of the information received by phone , the clhg coordinator started the procedure that is in place for patients in severe hypothermia , with the option of extracorporeal warming . he notified the team on duty at the cardiac surgery clinic of the john paul ii hospital in krakow , and an operating theater , as well as ecmo equipment were booked . due to poor atmospheric conditions ( bad weather ) , it was not possible to use an emergency helicopter . the clhg coordinator and the dispatch center in krakow arranged ambulance transport with a doctor for the patient suspected to have ii / iii stage hypothermia . transport was to take place from the scene of the incident directly to the john paul ii hospital . the decision was made to supply the ambulance with extra equipment ( i.e. , a device for mechanical chest compression and a low - temperature thermometer , both of which were taken from another emergency service ambulance working in the vicinity ) . ht , the coordinator for extracorporeal treatment of severe hypothermia patients took the medical history of the victim from his family . information about the lack of allergies and chronic illnesses and the medication taken was obtained . at 23:05 the rescuers reached the place where the emergency medical rescue team were waiting . after being carried into the warmed ambulance and having the monitoring devices connected , at 23:11 the patient suspected of hypothermia ( ht ) the patient was intubated , intravenous access was obtained and the transfusion of warm infusion fluids was implemented . the clhg coordinator advised the team managing the transport not to undertake advanced cardiovascular life support ( acls ) procedures there and told them to start transporting the patient to hospital as quickly as possible . at 23:26 the team set off with the jpii hospital as their destination . thanks to the continuous cooperation with the medical rescue team , the coordinator was able to evaluate the course of the acls procedures undertaken during transport . at 23:35 there was a brief period of agonal cardiac rhythm with pea ( pulseless electrical activity ) ( fig . the team covered 98 km by land . throughout the journey , the clgh coordinator had current information about the location of the emergency medical rescue team and the expected time of arrival at the destination hospital . the patient had asystole and anisocoria ( the left pupil was larger than the right one ) . with the emergency rescue team , and in urgent mode , he was taken to the already prepared operating theater , where he arrived at 1:15 . after cannulation of the femoral artery and femoral vein , arteriovenous extracorporeal membrane oxygenation ( ecmo ) was started at 1:35 . 200 j defibrillation was attempted , achieving the return of spontaneous circulation , but due to the persisting symptoms of cardiogenic shock , ecmo was continued until the morning hours of 1:01:2015 ( duration of ecmo therapy32 hours ) . the duration of circulatory arrest , from the time of diagnosis to administering extracorporeal circulation was 150 minutes . arterial blood gas values , blood glucose , hemoglobin and blood chemistry levels the patient regained consciousness and was extubated on january 2 , 2015 , and left the icu on january 7 , 2015 in a good neurological status ( glasgow coma scale 15 , cerebral performance category 1 ) , without anisocoria . at the time of leaving the hospital , the echocardiogram showed that the patient had a normal left ventricular ejection fraction ( ef-65% ) . prognosis in circulatory arrest in the course of accidental hypothermia is surprisingly good , despite even long periods of hypoperfusion ( dunne , 2014 ) . such treatment results are possible mainly owing to the cold protection of the brain , maintaining continuous organ perfusion obtained by means of manual or mechanical chest compression , and using extracorporeal warming ( brown et al . , 2012 ; dunne et al . , 2014 ; meyer et al , 2014 ; paal and brown , 2014 ; zafren et al . , 2014 ) . the guidelines proposed by gordon et al . ( 2015 ) suggest to start immediate continuous cardiopulmonary resuscitation ( cpr ) , to minimize interruptions and apply mechanical chest compressions as soon as possible , in circulatory arrest due to primary severe hypothermia . delayed or intermittent cpr may be considered only when continuous cpr is impossible , particularly during difficult evacuations . in the described case , the decision was made to supply the ambulance with extra equipment ( i.e. , a device for mechanical chest compression and a low - temperature thermometer , both of which were taken from another emergency service ambulance working in the vicinity ) . mechanical compression does not have a survival advantage over manual compression , but manual compression can not be done safely or effectively in a moving ambulance . the fourth element , which can significantly impact the prognosis is the appropriate organization of the rescue operation . good coordination , planning , foresight , and acting in accordance with the agreed procedures in the rescue scheme , makes it possible to shorten the time of reaching the destination hospital and effective treatment ( gordon et al . , 2014 ) . the case described here is an example of optimal management on the part of the coordinator and provides proof that the system that had been elaborated works in an effective way . notifying the coordinator of a search operation is one of the more important elements of the procedures in the rescue scheme under discussion . according to the algorithm that is in place , mountain rescue services ( 6 units of mountain rescue services covering the area of 17 680 km ) are obliged to give notification of rescue operations , especially ones conducted in conditions when there is a risk of the occurrence of hypothermia . thanks to this , it is possible to start preparations early . what is equally significant is the regular exchange of information between the coordinator and the units conducting the search and rescue medical operations . in the case described here , the coordinator had complete information about the victim transmitted to him by mountain rescuers , and was given all the parameters of the acls procedures undertaken during the ambulance transport . the immediate provision of the device for mechanical chest compression , which was taken aboard the ambulance transporting the patients as extra equipment , proved how excellent the planning and foresight of the operation was . on the basis of the information gathered , we have created a regularly updated map of the equipment at the disposal of hospitals and medical emergency services , so that coordinators can use them as required . cooperation with the dispatch center in krakow , which has agreed to the procedures in the rescue scheme elaborated by the clhg , is a very important aspect of such operations . in the case described here , on the basis of the information obtained , the coordinator made the decision that there was a high likelihood of circulatory arrest occurring , and therefore made arrangements for the device to be waiting for the patient at the place where he was taken over from the mountain rescuers . general knowledge about the procedures and treatment in managing such cases can therefore be incomplete and out of date . we confirmed that in our recent survey among 42 ers in poland providing emergency healthcare for the population of 5,305,000 ( kosinski et al . , 2015 ) . we believe that in the case being described , this element ( role of coordinator ) could also have influenced the outcome . it is , however , worth noticing that thanks to continuous information campaigns and regular training , the procedures in the rescue scheme that were developed are more and more universally recognized and used . knowledge of the principles of proceeding in hypothermia must be generally known , especially in the mountain rescue community . the recommendations and guidelines elaborated by icar and erc are therefore invaluable because they provide the basis for developing local protocols ( soar et al . , 2010 ; durrer et al . , the case described here confirms that it is possible to achieve full recovery of neurological functions in patients with circulatory arrest in the course of severe hypothermia , even after resuscitation procedures taking a few hours . at the same time , we have proven the effectiveness of the system that had been elaborated , thanks to which patients are referred to specialized hypothermia centers by means of a precisely pre - arranged route and set procedures . for obvious reasons , it is not possible to develop an ideal system , one in which all the possibilities can be foreseen . in another mountain rescue operation supervised by a clhg coordinator , some delays were unavoidable , and resuscitation procedures before introducing ecmo support in the destination hospital took 5 hours and 45 minutes ( own , unpublished data ) . however , similarly to the case fully described in that article , a full recovery of neurological functions was achieved . the element that could have been improved in this case was the delayed measurement of core temperature . neither the mountain rescuers , nor the emergency medical rescue team had appropriate equipment to perform this . core temperature ( tc ) measurement is the only diagnostic tool to assess the severity of hypothermia accurately . early tc measurement can significantly influence the course of rescue and medical proceedings and is by all means recommended ( strapazzon et al . , , the optimal thermometer should be minimally invasive , easy to handle , and independent of environmental conditions . epitympanic temperatures were comparable to invasive tc measurements in cases of deep hypothermia , however there is no clear evidence for the reliability of epitympanic tc at low ambient temperatures . every new case of hypothermia provides a topic for discussion to us and an incentive to introduce modifications raising the effectiveness of the system that had been introduced . its basic elements will always be the knowledge of the team comprising the rescue units , the cooperation of the units taking part in the operation , and good coordination of activities . good coordination , planning , predicting possible problems , and acting in accordance with the agreed procedures in the scheme , make it possible to shorten the time of reaching the destination hospital and implement effective treatment . the severe accidental hypothermia center was set up in krakow in 2013 in order to improve the effectiveness of the treatment of patients in the advanced stages of severe hypothermia .

atypical haemolytic uraemic syndrome ( ahus ) is a rare disease characterized by haemolytic microangiopathic anaemia , thrombocytopaenia and acute onset of renal failure , in the absence of escherichia coli infection . renal damage usually progresses to end - stage renal disease ( esrd ) , sometimes being accompanied by signs of extrarenal thrombotic microangiopathy ( tma ) . we report a case of full neurological and haematological recovery after eculizumab treatment in a patient with esrd secondary to chronic ahus refractory to plasmatherapy while she was under dialysis . it highlights the use of eculizumab for controlling extrarenal manifestations of ahus in this population . abstractdarocha , tomasz , sylweriusz kosinski , maciej moskwa , anna jarosz , dorota sobczyk , robert galazkowski , marcin slowik , and rafal drwila . the role of hypothermia coordinator : a case of hypothermic cardiac arrest treated with ecmo . high alt biol med 16:352 - 355 , 2015.we present a description of emergency medical rescue procedures in a patient suffering from severe hypothermia who was found in the babia gora mountain range ( poland ) . after diagnosing the symptoms of ii / iii stage hypothermia according to the swiss staging system , the mountain rescue service notified the coordinator from the severe accidental hypothermia center ( clhg ) coordinator in krakow and then kept in constant touch with him . in accordance with the protocol for managing such situations , the coordinator started the procedure for patients in severe hypothermia with the option of extracorporeal warming and secured access to a device for continuous mechanical chest compression . after reaching the hospital , extracorporeal warming with ecmo support in the arteriovenuous configuration was started . the total duration of circulatory arrest was 150 minutes . the rescue procedures were supervised by the coordinator , who was on 24-hour duty and was reached by means of an alarm phone . the task of the coordinator is to consult the management of hypothermia cases , use his knowledge and experience to help in the diagnosis and treatment . and if the need arises refer the patient for ecmo at clhg . good coordination , planning , predicting possible problems , and acting in accordance with the agreed procedures in the scheme , make it possible to shorten the time of reaching the destination hospital and implement effective treatment .

involutional entropion is a common condition in asian countries , including japan.1 one cause of involutional entropion is weakening of the capsulopalpebral fascia ( cpf).2 the aged , thin , membranous nature of the cpf limits the results of correction by the original jones procedures alone , so we added the modified hotz procedure to the entropion repair . our modified hotz procedure binds the dermis to the tarsus and corrects the ciliary orientation . to determine the efficiency of the additional hotz procedure , we compared the recurrence rates and operation times in corrections performed with and without this additional procedure . from april 2010 to december 2011 , one surgeon performed lower - lid surgeries using jones cpf - tightening procedure with the addition of the modified hotz procedure ( ciliary - everted suture ) . fifteen patients ( six male and nine female ) underwent this combined procedure for a total of 21 eyes ( six right eye only , three left eye only , six both eyes ) . from january 2010 to september 2010 , the same surgeon performed the jones procedure alone on eight patients ( four male and four female ) for nine eyes ( six right eye only , one left eye only , one both eyes ) . after injection of 1% xylocaine with 0.01% epinephrine , the lower - eyelid skin pinch is cramped . a horizontal skin incision is made 5 mm below the eyelash line , and the orbicularis under the skin is exposed . the layer of the orbicularis is identified by 4 - 0 silk - suture traction . the portion of the orbicularis over the tarsus is removed , and the layer between the orbicularis and the septum - cpf complex is separated . prior to dissection of the posterior cpf layer , the cpf complex is cut at tarsal height , and the conjunctiva is visible at the bottom layer of the lower eyelid . between the cpf and conjunctiva , a sparse red tissue layer resembling the mller muscle sometimes exists this is thought to be the posterior layer of the cpf . without puncturing the conjunctiva , next , the orbital fat layer beneath the septum is identified , and the layer is dissected inferiorly approximately 78 mm from the tarsus ( figure 1b ) . the anterior cpf layer exists just under the fat layer . after the exposure of the anterior layer , an advancement of approximately 5 mm ( tarsus - cpf tightening ) is performed with three mattress sutures of 6 - 0 polysorb ( figure 1c and d ) . after the jones procedure with cpf is completed , the modified hotz procedure ( ciliary - everted suture ) is performed between the tarsus and solid dermis tissue ( figure 1e ) . the ciliary directions are shown to be corrected after the operation ( figure 1f ) . figure 2 shows the cpf advancement on the tarsus ( green line ) and the hotz rotation suture ( blue line ) . the average age of two groups was 76.4 years , with an age range of 6685 years . the cases in which the jones procedure alone was used ( average age 76.0 years , nine eyes total ) reported three recurrences , which corresponds to a recurrence rate of over 30% at a follow - up duration of at least 6 months . the recurrent cases required an eyelid margin split or a lateral tarsal split for additional correction . the cases in which the combination method was used ( average age 76.7 years , 21 eyes total ) reported two cases of complications ( one recurrence and one ectropion ) , which corresponds to a recurrence rate of 5% . the one instance of recurrence occurred 9 months after surgery and required an eyelid margin split and an additional hotz procedure for correction . the instance of ectropion presented with severe horizontal laxity before surgery that developed into ectropion 3 months after surgery . although the direction of the cilia was corrected , this eyelid required a lateral tarsal strip ( lts ) . the jones procedure used an eyelid pinch and required an average of 22.6 minutes to complete ; the combined method required an average of 33.4 minutes to complete . the jones procedure was first reported in 1963.3 however , this procedure was not popular in japan until it was disseminated by kakizaki et al.4 the jones procedure tightens the cpf by plication , thus reinforcing the vertical traction power of the lower - lid ligaments . however , the cpf often presents as a thin and membranous structure in aged patients and is not strong enough to correct the lower - lid direction permanently . kakizaki recommended an anterior posterior double - layer advancement by means of this weakness.5 another study has reported rates of recurrence of 7%15% after cpf plication.6,7 the jones procedure in our hospital resulted in a higher recurrence rate of 30% , indicating that this an unreliable procedure . the surgical videos showed that the former case had a slightly thin cpf and the latter one had a slightly thick cpf . however , the direction of the cilia at the end of the surgeries was not different between these cases . thus , cpf thickness is not an appropriate tool for making decisions about additional procedures . the vertical laxity is corrected by the jones procedure , and the horizontal laxity is corrected by the lts.8 however , the lts is not an easy technique , and the operation is painful and bloody . the wheeler procedure is a more popular technique for horizontal plication of the eyelid orbicularis.9 however , the utility of this procedure is also decreased by the aging process , and the experimental recurrence rate is high.10 moreover , the original jones procedure removes the orbicularis situated over the tarsus and is incompatible with the wheeler procedure . previous studies , including our own , report recurrence rates of less than 10% in congenital entropion patients.11,12 jones et al introduced a modified procedure 10 years after the establishment of the original technique . his modified method tied skin cpf skin.13 in 1974 , schaefer recommended connecting the lid skin to the tarsus by taking a bite through the lower edge of the skin muscle.14 we selected dermis just under the side of the cilia to tarsus suture , which is a relatively solid tissue - binding site . this combination method results in a significantly higher success rate than when the jones procedure alone is used . the p - value was 0.034 , as calculated by the chi - square test for independence . a 5% recurrence rate is a much more desirable result compared to the 30% recurrence rate resulting from application of the simple procedure . the additional hotz procedure requires only 10 additional minutes in surgery , making it one of the best choices among the procedures discussed here . we will now perform this combined procedure for all involutional entropion cases , following a half - year transitional phase . because no technique achieved a zero rate of recurrence , additional steps taken to correct the cases of recurrence are outlined . when the cilia are located too close to the eyelid margin , we perform the margin split procedure . when the lower eyelid exhibits too much laxity and requires horizontal correction to increase its strength , we add lts to the lateral orbital periosteum . subsequent to this procedure , we perform ciliary electrolysis or the wojno ciliary removal procedure.15 it is essential to have a variety of additional techniques available to correct issues of recurrence in the treatment of entropion . pulmonary embolus ( pe ) refers to obstruction of the pulmonary artery or one of its branches by material ( e.g. , thrombus , tumor , air , or fat ) that originated elsewhere in the body . despite the high - incidence of pulmonary embolism , its diagnosis continues to be difficult primarily because of the nonspecific nature of the presenting signs and symptoms . the most common sources of pulmonary embolism are the pelvic veins or deep veins of the thigh . hemodynamically unstable pe results in hypotension , which by definition is systolic blood pressure < 90 mmhg for a period > 15 min or that requiring vasopressors or inotropic support and not explained by other causes including sepsis , arrhythmia , left ventricular dysfunction from acute myocardial ischemia or infarction , or hypovolemia . hemodynamically stable pe is defined as pe that does not meet the definition of hemodynamically unstable pe . majority of the cases of pulmonary embolism can be managed with medical therapy alone . for more than 30 years , thrombolytic agents have been used to dissolve or reduce the embolus and improve the circulation . however , embolectomy is indicated in patients with hemodynamically unstable pe in whom thrombolytic therapy is contraindicated . it is also a therapeutic option in those who fail thrombolysis and surgical intervention may be indicated in certain instances . currently , only few indications for surgical embolectomy have been described which include contraindications to thrombolytic therapy , presence of persistent thrombi in the right heart or pulmonary arteries , or deteriorating hemodynamic status . we report two cases of acute massive pe , which was managed successfully with surgical embolectomy . electrocardiogram ( ecg ) showed sinus tachycardia , right axis deviation , and right bundle branch block . routine investigations were normal with erythrocyte sedimentation rate ( esr ) 90 mm 1 h westergren . two - dimensional ( 2d ) echocardiography revealed dilated right atrium / right ventricular ( ra / rv ) , severe tricuspid regurgitation ( tr ) with pulmonary artery systolic pressure of 70 mmhg , and clot in rv apex and right pulmonary artery ( rpa ) [ figure 1a and b ] . the patient was subjected to surgical embolectomy with rv and rpa clot extraction [ figures 2a , b and 3 ] . ( a ) two - dimensional echo apical four chamber view shows clot in right ventricular apex and ( b ) : two - dimensional echo short axis shows thrombus in the right pulmonary artery ( a ) extraction of the right ventricular clot . ( b ) extraction of the thrombus in right pulmonary artery right ventricular and right pulmonary artery thrombus a 65-five - year - old male patient presented with acute onset of dyspnea on the 15 postoperative day for traumatic rupture of urinary bladder . he is a k / c / o hypertension with chronic obstructive pulmonary disease . on examination , routine investigation revealed hemoglobin of 12 g% , esr - 70 mm 1 h westergren . 2d echocardiography revealed dilatation of ra , rv , rpa ostium , and ra fuzzy hypoechoic lesion [ figure 4 ] . computed tomography pulmonary angiogram revealed pulmonary thrombus in proximal rpa and normal main pulmonary artery [ figure 5 ] . in view of recent surgery for traumatic rupture of urinary bladder and clot in ra , thrombolysis was not considered , and the patient was subjected to surgical embolectomy [ figure 6 ] . two - dimensional echo short axis shows thrombus in the ostium of right pulmonary artery computed tomography pulmonary angiography shows thrombus in the right pulmonary artery extraction of thrombus in the right pulmonary artery massive pe is defined as rv dysfunction or a systolic blood pressure < 90 mmhg or a drop in systolic blood pressure of 40 mmhg from baseline for a period > 15 min . in patients with massive pe , 50% patients die within 30 min , 70% die within 1 h , and more than 85% die within 6 h of the onset of symptoms . clinically , diagnosis is difficult due to the nonspecific nature of the symptoms and signs of pe and investigations including routine blood , arterial blood gas measurement , electrocardiography , and chest radiography , however all lack specificity ; thereby limiting their utility in reaching a definitive diagnosis . the differential diagnosis for pe is many which include pneumothorax , pleuritis , pneumomediastinum , pericarditis , acute myocardial infarction , aortic dissection , esophageal rupture , lung cancer , rib fractures , and metastatic deposits . the most common sources of pulmonary emboli are the pelvic veins or deep veins of the thigh . pulmonary arterial obstruction by clot causes platelets to release vasoactive agents such as serotonin , which may lead to further elevation of pulmonary vascular resistance . this redistribution of blood flow increases alveolar dead space with a resultant ventilation / perfusion mismatch that impairs gas exchange . right ventricular after load increases , tension rises in rv wall and may lead to dilatation , dysfunction , and ischemia of right ventricle . in the absence of absolute contraindications , thrombolysis is the indicated treatment for patients with acute massive pe , who present with shock and is reported to achieve good outcomes . in those patients whose condition worsens despite thrombolysis , surgical intervention should be strongly considered . currently , only a few indications for surgical embolectomy have been described which include contraindications to thrombolytic therapy , presence of persistent thrombi in the right heart or pulmonary arteries , or deteriorating hemodynamic status . early surgical intervention may be associated with a survival benefit in such situations . in one nonrandomized case series of patients with massive pe complicated by shock the main predictor of mortality in patients undergoing surgical pulmonary embolectomy is preoperative cardiac arrest requiring resuscitation . patients with hemodynamic collapse prior to surgery have an operative mortality ranging from 43% to 84% . massive pe requires immediate therapy to restore pulmonary arterial flow . in the absence of treatment , anticoagulation is the standard of care for pe , with the addition of thrombolysis or surgical embolectomy when the clot burden and clinical presentation warrant more aggressive treatment . surgical embolectomy in hemodynamic instability due to acute pe is reserved for patients in whom thrombolysis ( systemic or catheter - directed ) is contraindicated , and is an option in those in whom thrombolysis has failed . however , surgical embolectomy may show favorable outcomes in cases such as echocardiographic evidence of an embolus trapped within a patent foramen ovale , or present in the ra , or right ventricle . surgical embolectomy , therefore , should not simply be considered as a treatment of last resort , but may warrant earlier consideration in certain conditions .

purposeinvolutional entropion is a common condition in asian countries , including japan . one cause of involutional entropion is weakening of the capsulopalpebral fascia ( cpf ) . the aged , thin , membranous nature of the cpf limits the results of correction by the original jones procedure ( cpf tightening ) alone , so we added the modified hotz procedure to the entropion repair . we then compared the recurrence rates and operation times in corrections performed with and without this additional procedure.casesfrom april 2010 to december 2011 , one surgeon performed lower - lid surgery using the jones procedure with the addition of the modified hotz procedure . fifteen patients ( a total of 21 eyes ) underwent this combined procedure . previously , the same surgeon performed the jones procedure alone for eight patients ( a total of nine eyes).resultsthe average age of the two groups was 76.4 years , with an age range of 6685 years . all cases reported acceptable ciliary orientation at the end of the surgery . however , patients who underwent the jones procedure alone ( nine eyes total ) reported three cases of recurrence after at least 6 months of follow - up . patients who underwent the combined procedure reported two complications : one recurrence and one ectropion . the recurrence rate was 5% . the jones procedure using eyelid pinch required an average of 22.6 minutes to complete ; the combined method required 33.4 minutes to complete.conclusionthe combined method resulted in a significantly higher success rate than the jones procedure alone ( p < 0.05 ) . the 5% failure rate of the combined method was found to be superior to the 30% recurrence rate of the jones procedure . as a result , the hotz procedure enhanced the results of the entropion correction and required only 10 additional minutes of surgery . we now perform this combined procedure for all cases . acute massive pulmonary embolism is a life - threatening emergency that must be promptly diagnosed and managed . over the last several years , the use of computed tomography scanning has improved the clinician 's ability to diagnose acute pulmonary embolism . we report two cases of acute massive pulmonary embolism who presented with sudden onset of dyspnea and underwent successful open pulmonary embolectomy . the first case presented with acute onset of dyspnea of 2 days duration , in view of hemodynamic deterioration and two - dimensional echocardiography , it revealed clot in right ventricular ( rv ) apex and right pulmonary artery ; the patient underwent cardiopulmonary bypass and open pulmonary embolectomy with rv clot extraction . the second case presented with a sudden onset of dyspnea on the 15th postoperative day for traumatic rupture of urinary bladder , in view of recent surgery , the patient was subjected to surgical embolectomy . following surgical intervention , both the patients made a prompt recovery .

the symptoms of malaria include fever and anemia , and most of the deaths are caused by the parasite plasmodium falciparum . the merozoite form of the parasite invades red cells , grows to form ring- , trophozoite- and schizont stages , and after rupture of the infected red cell new merozoites are released that are ready to enter uninfected red cells . merozoite invasion is a process that takes only a few minutes , but it involves several complex receptor - ligand interactions . initial attachment of the merozoite is mediated by merozoite surface proteins such as msp1 and msp2 , and is followed by reorientation of the merozoite where apical membrane antigen 1 ( ama1 ) is of importance [ 4 , 5 ] . other ligands such as erythrocyte - binding antigens ( ebas ) , for example , eba140 , eba175 , and eba181 and p. falciparum reticulocyte - binding homologues ( pfrhs ) , including pfrh1 , pfrh2 , pfrh4 , and pfrh5 have also shown to be involved in the invasion process [ 69 ] , even though the exact function of each antigen is not known . genetic polymorphisms exist for many of the above - mentioned ligands , and based on some genes like msp2 , parasites can be grouped into two major allelic types : 3d7 and fc27 . serine repeat antigens ( seras ) are proteases that take part in forming a protein complex that is associated with the merozoite surface [ 1012 ] , and entry into the red blood cell is finally completed by an actin - myosin motor movement [ 13 , 14 ] . individuals who live in malaria endemic areas eventually develop immunity , but only slowly and after repeated exposure [ 15 , 16 ] . women have a greater risk of succumbing to malaria , and the fetus is also at risk . it is known that antibodies are important in the defence against malaria , and it has been shown that passive transfer of antibodies from immune donors to individuals with p. falciparum infection reduces parasitemia and clears clinical symptoms [ 1821 ] . however , exactly which specific antibodies are protective against future disease are not yet defined , and how they should be measured is even less clear . this information is urgently needed to be able to develop a functioning vaccine , something that has so far failed . we will here discuss the pros and cons for different methods available such as elisa , invasion inhibition assays ( iias ) , antibody - dependent cellular inhibition ( adci ) , and affinity , and we compare it to measurements of antibodies in other diseases and how the overall evaluation of immunity or vaccine status of malaria could possibly be improved . when antibodies directed against different p. falciparum antigens have been measured , elisa has usually been the method of choice . in this static method , proteins are coated to a plate and levels of antibodies in plasma from patients with ( or without ) malaria can be estimated . when recombinant proteins are used , only antibodies directed against specific antigens are analyzed . in real life , the antigen of choice is located together with several other antigens , for example , on the merozoite surface , and there might be interaction or competition between binding of different antibodies , something that is not accounted for in an elisa . it has , for example , been shown for msp1 that there are blocking antibodies that can compete with the binding of cleavage - inhibiting antibodies for epitopes on the merozoite [ 22 , 23 ] , and there are also other studies that have demonstrated that mixing of different antibodies can influence the outcome of the assay . this kind of studies indicates that we should more look at using assays where the function of antibodies is studied , but the reason for why elisas are continued to be used to such a major extent is probably that they are very easy , fast , and robust to perform compared to functional assays . when recombinant proteins are applied in elisas , the result might depend on which part of an antigen that is selected for use in the assay . for msp1 , it has been shown that antibodies against msp1 - 19 were associated with some protection , while antibodies against msp1 block-1 were not . however , even when the same subdomain has been used such as in studies of eba175 , contradictory results have been achieved for whether there was a protective effect of antibodies or not [ 26 , 27 ] . when red cells burst due to egress of merozoites , a lot of debris will be left in the blood stream that needs to be removed , and many of the antibodies might help in doing this but this does not mean that the antibodies will protect from future disease . if only elisas are used , it is difficult to discern which antibodies are functionally important . in general , higher levels of antibodies are found in elisas in older individuals in endemic areas , while lower levels of antibodies are seen in younger individuals in the same areas . this was recently shown for the ebas for example and it has been shown earlier for other antigens as well [ 7 , 26 , 27 , 29 , 30 ] . however , even though an individual has high levels of antibodies , they can still develop malaria , and an individual with relatively low levels of antibodies can be fully protected from clinical and severe malaria [ 25 , 3152 ] . in vaccine trials , antibodies measured by elisa have been shown to often be short - lived , and most patients will still get malaria in spite of presence of antigen - specific antibodies . from a population perspective , elisas can be used to make an overall estimation of how much exposure there has been to malaria , but for each individual it is not possible to make an exact determination of the immune status . the only thing that can for sure be concluded from a positive response in elisa is that the individual has at some stage during his / her lifetime been exposed to malaria . when elisas against different antigens are combined , more information can possibly be acquired about the level of immunity in investigations of the breadth of antibodies , but the question of whether it is just a measure of exposure will still remain . a way of improving the elisas would be to more often use standardized controls , allowing for the measurement of exact amounts of specific antibodies instead of titers . in conclusion , elisas are easy and robust to perform , and they can clearly give us information about whether or not an individual has ever been exposed to malaria . with combinations of different antigens and standardization of the assays , more information can possibly be provided . however , elisas do not tell us anything about the function of the antibodies , and on an individual level , elisas will not give us complete information about immunity . one assay that has been used to try and better determine the function of antibodies in plasma is growth inhibition assay or invasion inhibition assay ( iia ) . antibodies directed against merozoite antigens are thought to function by directly inhibiting invasion of new red cells , which will then stop further multiplication of parasites , or through adci . by adding immune plasma , which contains antibodies to growing parasites , the inhibitory function of the antibodies can be evaluated in comparison to parasites where no plasma has been added . the downside of iias compared to elisas is that they are much more labor intensive , but on the other hand all proteins are expressed in their native environment and many both known and unknown potential interacting factors are included in the assay . there have been several studies that have shown invasion inhibitory activity of antibodies from human plasma , both when total igg has been used and when malaria - antigen - specific fractions have been used [ 22 , 5458 ] . some studies have shown increasing invasion inhibition with age while others have shown more invasion inhibition in children [ 56 , 60 ] . this kind of contradictory results might be explained by different functions in the antibodies repertoire being important during development of immunity , compared to when immunity is already established , but it might also mean that the assay is not yet fully optimized to show who is immune or not . one attempt to improve the iia is by adding monocytes ( adci ) . when adci has been employed , some antigens like msp3 and glurp [ 61 , 62 ] have shown an inhibitory effect only when monocytes were included in the iia . however , with adci there seems to be a major variability in the assay that can be seen from day to day even using the same donor of monocytes , making it difficult to standardize the assay . if this assay could be improved and standardized , it might add very valuable information about different antibodies . another attempt to try and improve iia is to use knockout lines of parasites . here , a single antigen can be studied in its natural environment and with the correctly folded protein , and a comparison can be made between the wild - type and the knockout parasites . this has been used , for example , for the ebas , where it was shown that antibodies against eba175 was responsible for a major part of the inhibitory activity in some individual plasma samples , while other samples seemed not to have any functional antibodies against this protein at all . this kind of results is important for selection of potential vaccine candidates , especially for ruling out those antibodies that have no effect . some antigens have been difficult to knock out , in which case other reagents might have to be added to the assay such as blocking agents , to find out which antibodies are causing the inhibitory effect . in conclusion , iias are labor intensive but can provide important information especially for comparisons between knockout and wild - type parasites , where the function of a single potential vaccine candidate antigen can be evaluated . another way of looking at antibodies against merozoite antigens is to study the affinity / avidity of antibodies . some studies have tried to use elisa with added nh4scn to evaluate affinity , but the results for this have been inconsistent [ 64 , 65 ] . a new way of investigating affinity for vaccine trials has opened up with methods like surface plasmon resonance ( spr ) [ 66 , 67 ] , where affinity of antibodies can be estimated under flow , something that ought to be more similar to the physiological situation compared to static assays . with this method , association and dissociation of antibodies binding to their target antigens this method has before mainly been used with monoclonal antibodies in malaria research , but it has recently been applied also to polyclonal antibodies . it has , for example , been shown that affinity of antibodies against ama1 increased with age , and the presence of high affinity antibodies in plasma against msp2 - 3d7 was associated with protection against malaria . in this study , most plasma samples showed a relatively rapid on - rate , indicating that whether the concentration of antibodies in the samples is high or low , the antibodies will still bind quite fast to their antigens . this is important for considering whether an antibody will function in inhibiting merozoite invasion or not , since invasion is a process that takes only a couple of minutes . the dissociation rate ( which is concentration independent ) might therefore be more important for whether an antibody will function or not . in the referred study , monoclonal antibodies were also used and it could be seen that some bound with so low dissociation rates , that a value for the dissociation rate could not be obtained . however , investigations of affinity of antibodies in malaria are yet a very new field which needs a lot more studies to be able to make firm conclusions , and standardized protocols needs to be in place to facilitate interpretation of the results . in other infectious diseases , such as bacterial diseases [ 69 , 70 ] , toxoplasma , or hiv , studies of affinity of polyclonal antibodies in malaria is a new field that could add a lot of information both about how immunity is formed and for vaccine trials , and more work in this area is needed . when different assays are used to evaluate antibodies against malaria , the methods often show results that do not correlate with each other . for example , spr has been shown to correlate with elisa for ama1 , which binds with relatively high affinity , but not for msp2 , which binds with lower affinity . when iia has been compared to elisa , some people have shown correlations while others have not seen any correlations [ 36 , 73 ] . this is probably because iia is a functional assay , while elisa only estimates the levels of antibodies . an example of the lack of major correlations between methods ( iia and elisa ) is shown in figure 1 . this illustrates the difficulties in estimating immunity against malaria . for vaccine trials , one has to be careful with interpretation of results , as the results could vary a lot depending on which method is used . in other fields such as hiv , international consortia have established common standards to be used in immunological assays , and this might be applicable for elisas , but more challenging for the functional methods yet available in malaria . none of the methods described here are good enough on their own to give a complete picture of an individual 's immune status . elisas are good at giving us information about whether any immune response at all is mounted against a potential vaccine candidate , and from a population perspective when many samples need to be analyzed , elisas are easy to perform . however , if we want to know something about the function of a specific antibody , iia with the usage of knockout and wild - type parasites should be the way forward . even though these assays are more cumbersome to perform , they will add valuable information . affinity of antibodies has so far been very scarcely studied in malaria research compared to many other diseases , and expansion of this field should add important information both for knowledge about immunity and for vaccine trials . more studies are needed that employ different methods together in the same patient cohorts to get a more full picture of which functions of antibodies are important during different stages of development of immunity against malaria . in conclusion , which method should be used depends on what we want to know about the antibodies . if we only want to know whether antibodies are formed or not , elisas can be used , but if we want to know something about the function of the antibodies , more elaborate assays such as iia have to be applied . to get the full picture about immunity status in an individual , the methods available have to be developed more and probably combined to a bigger extent , but with new methods such as those available for affinity measurements there is hope that this situation can be improved . ocular cysticercosis is caused by the growth of the larval form of taenia solium within ocular tissues.1,2 the cysts may be located in descending order of frequency in the subretinal space ( 35% ) , vitreous ( 22% ) , conjunctiva ( 22% ) , anterior segment ( 5% ) and orbit ( 1%).3 intraocular cysticercosis usually presents with reduced vision and ocular inflammation . it is believed that the larva reaches the subretinal space through the posterior ciliary arteries.3 - 6 as the cyst develops , it may cause exudative retinal detachment.6 as long as the cyst remains viable , it evokes little or no inflammatory response . once the cyst starts degenerating , an antigen , which may be a metabolic by - product or toxin , leaks from the cyst and induces an inflammatory reaction , manifesting as vitritis , uveitis and sometimes endophthalmitis.3 - 6 most often , the characteristic intraocular cyst can be visualized by indirect ophthalmoscopy or detected by b - scan ultrasound in the presence of hazy media.3 b - scan ultrasonography will show a curvilinear echo corresponding to the cyst wall together with an eccentric hyperechoic dot suggestive of the scolex . a - scan analysis reveals two high amplitude echoes representing the anterior and posterior walls of the cyst . mahendradas et al7 highlighted the features of intraocular cysticercus cyst employing spectral domain oct ; they clearly delineated the hyper - reflective wall of the subretinal cyst with a more hyper - reflective portion within its wall suggestive of the scolex / larva . cns involvement can be observed in approximately 90% of patients with ocular cysticercosis and mri is superior to computed tomography ( ct ) in detecting lesions of neurocysticercosis ( ncc).8 in addition , mri better illustrates cystic lesions in the base of the brain , cerebrospinal fluid ( csf ) spaces as in ventricular ncc and cisternal ncc , and also with intramedullary lesions.9 the scolex may be more readily apparent on mri than on ct . on mri , contents of live cysts ( vesicular stage ) are isointense relative to csf on t1- and t2-weighted images.10 treatment of ocular cysticercosis is mandatory since it has been reported that 80% of untreated cases result in severe ocular damage.3 antihelminthic drugs such as praziquantel or albendazole are effective in central nervous system and skin cysticercosis . . surgical removal of the cyst can also be performed through transretinal or trans - scleral routes.11 systemic corticosteroid coverage is required before and after surgical removal of the cysticercus.3

immunity against malaria develops slowly and only after repeated exposure to the parasite . many of those that die of the disease are children under five years of age . antibodies are an important part of immunity , but which antibodies that are protective and how these should be measured are still unclear . we discuss the pros and cons of elisa , invasion inhibition assays / adci , and measurement of affinity of antibodies and what can be done to improve these assays , thereby increasing the knowledge about the immune status of an individual , and to perform better evaluation of vaccine trials . a 31-year - old man of asian descent presented with loss of vision in his right eye from 6 months earlier . best corrected visual acuity ( bcva ) was limited to light perception in the right eye and was 6/6 , n6 in the left one . slit lamp examination revealed normal anterior segments bilaterally . intraocular pressure was 8 and 14 mmhg in the right and left eyes , respectively . fundus examination in the right eye showed a large subretinal cysticercus cyst , accompanied by total retinal detachment ( rd ) , severe extensive subretinal fibrosis and membranes ( fig . 1a).b - scan ultrasound ( alcon ultrascan , alcon laboratories , fort worth , texas , usa ) images illustrated total rd with a hyperechoic area within the cystic cavity suggestive of cysticercus scolex ( fig . 1b ) . spectral domain optical coherence tomography ( oct ) ( topcon 3d oct-2000 , topcon medical systems , oakland , new jersey , usa ) demonstrated a highly reflective cyst wall and a more hyper - reflective dome - shaped structure within the wall suggestive of the scolex ( fig . 1c ) . the posterior extent of the cyst could not be visualized due to its large size . t1-weighted contrast - enhanced magnetic resonance imaging ( mri ) of the brain demonstrated a ring - shaped enhancing lesion in the left cerebellar hemisphere with perilesional brain edema suggestive of neurocysticercosis ( fig . 1d ) . the patient was referred to a neurophysician and received a three month course of oral albendazole and steroids . eventually , he was recommended for follow - up care .

the online version of this article ( doi:10.1007/s13555 - 015 - 0088-z ) contains supplementary material , which is available to authorized users . dermoscopy is a valuable noninvasive technique that increases diagnostic accuracy in melanoma and non - melanoma skin cancer [ 1 , 2 ] . recently , its use as an aid in the monitoring of topical treatment response has also been described [ 24 ] . basal cell carcinoma ( bcc ) is the most frequent type of skin cancer in humans . surgical excision is still considered the gold - standard of treatment . however , a number of topical therapies are now available for the treatment of different types of basal cell carcinoma . despite the fact that bcc is associated with a low mortality and very rarely gives rise to metastatic disease , delays in its correct diagnosis and treatment prolong patient morbidity and increase the cost of care . dermoscopy has also shown to be useful in the assessment of early recurrence or tumor persistence . dermoscopic criteria such as pigmented structures , ulceration and arborizing vessels have been suggested to predict the presence of residual disease ( residual disease - associated dermoscopic criteria ) [ 4 , 5 ] . this case report exemplifies the usefulness of dermoscopy in the assessment of residual disease after incomplete surgical excision and also in the monitoring of topical treatment response . an otherwise healthy , 92-year - old woman was seen for the evaluation and treatment of a long - standing nodular lesion on the tip of her nose . the lesion was characterized as a solitary , 5 mm , smooth , nodular , pearly tumor with telangiectasia ( fig . 1 ) . dermoscopy confirmed the diagnosis of nodular bcc because of the presence of telangiectasia and arborizing vessels ( fig . 2 ) . the wound healed by secondary intention with daily topical application of a pirfenidone gel ( fig . 3 ) ( kitoscell - q , cell therapy and technology , s.a . de c.v . , mexico ) . histopathologic examination revealed a neoformation arising from the epidermis proliferating into the papillary and reticular dermis . it was constituted by an epithelial tumor with basaloid - appearing cells disposed in palisade at the periphery of the tumor nests . the tumoral nests were surrounded by a lymphocytic inflammatory infiltrate and a fibromyxoid stroma with stromal retraction artifact in certain areas . the tumor extended into the deep margins where tumoral nests were smaller , with angular shapes and interspersed by a more fibrous stroma . histology confirmed the diagnosis of infiltrating nodular bcc and remains of tumoral cells within the wound bed and deep margins ( fig . six weeks after the incomplete surgical excision , the esthetic result was acceptable ( fig . however , dermoscopy confirmed the persistence of the tumor characterized by a large arborizing vessel ( fig . for this reason and due to the ease of at - home application , daily topical treatment with 5% imiquimod cream for 8 weeks was started ( fig . clinically and dermoscopically the patient revealed an appropriate response to the topical treatment characterized mainly by erythema and mild ulceration ( fig . four weeks after the end of treatment , the clinical esthetic result was acceptable ( fig . 9 ) . at 12 months after the end of treatment dermoscopy confirmed the absence of any signs of recurrence ( fig . the patient was last seen 23 months after the end of treatment without any signs of recurrence and dermoscopy confirmed the eradication residual disease ( fig . all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1964 , as revised in 2013 . 1the lesion on the nose was characterized as a solitary , 5 mm , smooth , nodular , pearly tumor with telangiectasiafig . 2dermoscopy confirmed the diagnosis of nodular basal cell carcinoma because of the presence of telangiectasia and arborizing vesselsfig . 3wound healed by secondary intention with daily topical application of a pirfenidone gelfig . 4 a ( 10 , 20 ) and b ( 40 ) . histology confirmed the diagnosis of infiltrating nodular basal cell carcinoma and remains of tumoral cells within the wound bed and deep marginsfig . 6dermoscopy confirmed the persistence of the tumor characterized by a large arborizing vesselfig . 7 a , b daily topical treatment with 5% imiquimod cream was startedfig . 8 a f clinically and dermoscopically the patient revealed an appropriate response to the topical treatment characterized mainly by erythema and mild ulcerationfig . 9four weeks after the end of 5% imiquimod treatment the clinical esthetic result was acceptablefig . dermoscopy shows no signs of tumor recurrence and confirms eradication of residual disease the lesion on the nose was characterized as a solitary , 5 mm , smooth , nodular , pearly tumor with telangiectasia dermoscopy confirmed the diagnosis of nodular basal cell carcinoma because of the presence of telangiectasia and arborizing vessels wound healed by secondary intention with daily topical application of a pirfenidone gel a ( 10 , 20 ) and b ( 40 ) . histology confirmed the diagnosis of infiltrating nodular basal cell carcinoma and remains of tumoral cells within the wound bed and deep margins six weeks after the incomplete surgical excision the esthetic result was acceptable dermoscopy confirmed the persistence of the tumor characterized by a large arborizing vessel a , b daily topical treatment with 5% imiquimod cream was started a f clinically and dermoscopically the patient revealed an appropriate response to the topical treatment characterized mainly by erythema and mild ulceration four weeks after the end of 5% imiquimod treatment the clinical esthetic result was acceptable one year later the patient had no signs of recurrence confirmed by dermoscopy clinical and dermoscopic appearance 23 months after the end of treatment . basal cell carcinoma is the most frequent cancer of the skin and its incidence is increasing worldwide . surgery , including mohs micrographic surgery , is considered the gold standard of treatment and the most effective in terms of recurrence rates . incomplete excision has been associated with various recurrence rates ranging from 26% to 67% and 50% of recurrences occur within the next 612 months . certain anatomic locations such as the nose and inner canthus , as well as certain histologic subtypes such as infiltrative and multifocal , have been associated as predictive factors for incomplete excision . in cases where the surgical intervention is unable to completely remove the tumor , adjuvant therapy such as a second surgical procedure , radiotherapy , photodynamic therapy and topical immunotherapy are used . imiquimod is a toll - like receptor-7/8 ( tlr-7/8 ) agonist that acts as a topical immune response modifier and has been reported as a non - surgical alternative for the treatment of bcc , particularly for the superficial subtype [ 7 , 8 ] . topical imiquimod 5% cream promotes a th-1 immune response aimed at enhancing the removal of neoplastic cells . recently , imiquimod 5% cream has been described as a novel alternative to reduce deregulated hedgehog ( hh)/gli signal , independent of tlr signaling . small molecule targeting of hh signaling by inhibiting the essential pathway effector smoothened has proven exceptionally efficient for the treatment of advanced and metastatic basal cell carcinoma . however , severe side effects , limited response rates , and rapid development of drug resistance may limit the therapeutic success of smo antagonists . this reflects the need for the identification of alternative and additional strategies repressing oncogenic hh signaling . imiquimod 5% may be one of such alternatives as a topical , safe and relatively easy to use drug for the treatment of different bcc subtypes as adjuvant or neoadjuvant therapy . our case report exemplifies that dermoscopy is a valuable diagnostic aid that also serves to assess tumor recurrence / residual disease and to monitor topical treatment response . it also adds further evidence to the use of imiquimod as an important topical neoadjuvant or adjuvant therapy for the treatment of bcc and residual disease in cases with incomplete surgical excision [ 10 , 11 ] . all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1964 , as revised in 2013 . this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made . the lens samples were removed from the manufacturer packaging , placed front ( convex ) surface down on a clean glass slide , cross - sectioned with a razor blade and mounted onto a lens holder for sem imaging with a hitachi model s-3400n sem at an iso 17025 accredited laboratory . the images were recorded at an acceleration voltage of 10 kv under variable pressure ( 3050 mpa ) operating mode and using a back - scattered electron detection . one 500 magnification image of each cross - section was obtained to show the entire width of the sample and the general location of the pigment particles . a 500 magnification ( and in some cases a 2,000 magnification ) image was taken in a top - down or bottom - up view . to measure the depth of the pigment particles , the uncertainty of dimensional sem measurements is about 10 per cent ( providing an estimated level of confidence of 95%).21 three to 12 lenses per lens brand were tested for each of the following seven lens types : camax color ( cc ) ; 1-day acuvue define ( 1dad ) ; one - day delight max 2 ( dm2 ) ; seed eye coffret uv ( eco ) ; freshlook one - day color ( flc ) ; naturelle daily disposable ( nat ) ; and ticon cosmetic daily ring black ( tc ) contact lenses ( table 1 ) . lens brands , manufacturers , region available , variants and powers tested listed in alphabetical order atomic force microscopic images were acquired for the seven lens types in fluid phase with a dimension icon afm from bruker nano under contact mode using sharp nitride lever probes ( snl-10 ) with a spring constant of 0.06 newton per metre . the area of lens under afm imaging and the afm tip were kept hydrated with a droplet of standard lens packing solution . within each lens brand , multiple lenses from each power tested rms roughness and peak - to - peak values were obtained by analysing three 20 m 20 m regions ( front curve , back curve , pigmented and non - pigmented ) of these images . four replicates of rms and peak - to - peak measurements were selected for each area within each lens at a given location or pigment status . thus , a total of 12 measurements were recorded for each lens at a given location or pigment area . data from rms and peak - to - peak responses were determined to be log - normally distributed . these responses were analysed separately using a generalised linear mixed model with a log - normal distribution . location ( front versus back curve ) , pigment area ( pigmented versus non - pigmented ) and lens brand were included as fixed effects . all two - way and three - way interactions between location , pigment and brand were also included as fixed effects . replicates nested within lens number and area number were modelled as random effects within each brand . least squares mean values and their corresponding 95% confidence intervals were estimated for each brand , location and pigment combination . results from the sem image analyses were used to determine the appropriate pair - wise comparisons between lens brands . pair - wise comparisons were made for each lens brand for pigment versus no pigment on the lens surfaces . in this study , since the analysis was completed on the log - scale , the afm estimates and their interval bounds were exponentiated to provide model - based estimates on the original , untransformed scale . since multiple comparisons were considered , a simulation adjustment for alpha was completed to control the the lens samples were removed from the manufacturer packaging , placed front ( convex ) surface down on a clean glass slide , cross - sectioned with a razor blade and mounted onto a lens holder for sem imaging with a hitachi model s-3400n sem at an iso 17025 accredited laboratory . the images were recorded at an acceleration voltage of 10 kv under variable pressure ( 3050 mpa ) operating mode and using a back - scattered electron detection . one 500 magnification image of each cross - section was obtained to show the entire width of the sample and the general location of the pigment particles . a 500 magnification ( and in some cases a 2,000 magnification ) image was taken in a top - down or bottom - up view . to measure the depth of the pigment particles , the uncertainty of dimensional sem measurements is about 10 per cent ( providing an estimated level of confidence of 95%).21 three to 12 lenses per lens brand were tested for each of the following seven lens types : camax color ( cc ) ; 1-day acuvue define ( 1dad ) ; one - day delight max 2 ( dm2 ) ; seed eye coffret uv ( eco ) ; freshlook one - day color ( flc ) ; naturelle daily disposable ( nat ) ; and ticon cosmetic daily ring black ( tc ) contact lenses ( table 1 ) . lens brands , manufacturers , region available , variants and powers tested listed in alphabetical order atomic force microscopic images were acquired for the seven lens types in fluid phase with a dimension icon afm from bruker nano under contact mode using sharp nitride lever probes ( snl-10 ) with a spring constant of 0.06 newton per metre . the area of lens under afm imaging and the afm tip were kept hydrated with a droplet of standard lens packing solution . within each lens brand , rms roughness and peak - to - peak values were obtained by analysing three 20 m 20 m regions ( front curve , back curve , pigmented and non - pigmented ) of these images . four replicates of rms and peak - to - peak measurements were selected for each area within each lens at a given location or pigment status . thus , a total of 12 measurements were recorded for each lens at a given location or pigment area . data from rms and peak - to - peak responses were determined to be log - normally distributed . these responses were analysed separately using a generalised linear mixed model with a log - normal distribution . location ( front versus back curve ) , pigment area ( pigmented versus non - pigmented ) and lens brand were included as fixed effects . all two - way and three - way interactions between location , pigment and brand were also included as fixed effects . replicates nested within lens number and area number were modelled as random effects within each brand . least squares mean values and their corresponding 95% confidence intervals were estimated for each brand , location and pigment combination . results from the sem image analyses were used to determine the appropriate pair - wise comparisons between lens brands . pair - wise comparisons were made for each lens brand for pigment versus no pigment on the lens surfaces . in this study , since the analysis was completed on the log - scale , the afm estimates and their interval bounds were exponentiated to provide model - based estimates on the original , untransformed scale . since multiple comparisons were considered , a simulation adjustment for alpha was completed to control the cc , dm2 , eco and tc lenses were found to have pigments on the back surface ( concave side ) of the contact lens , while the pigments for nat and flc were found on the front surface ( convex side ) of the contact lens . the pigment particles were visible on the lens surface in top - down / bottom - up views , as well as in cross - sectional view appearing to extend from an average depth of 1.3 m to 3.5 m up to and including the lens surface for a depth range of 0.0 to 4.3m . the pigment particles for the 1dad were buried below the front surface of the lens at an average depth of 8.1 m with a range of 7.6 to 9.1 m ( table 2 ) . depth range for pigments from the lens surface when imaged by scanning electron microscope ( sem ) for each lens type 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable figures 1 7 show the cross - sectional depths at 2,000 magnification for each of the lens types . the images show no pigment particles on the top of the 1dad lenses , while a particle layer is on the surface of all other lenses tested . these results were used to determine the statistical comparisons to be completed for the afm findings ( surface pigmented lenses versus non - surface pigmented lenses ) . cross - sectional view at 2,000 magnification showing pigment enclosed within the lens matrix ( lm ) below the front lens surface ( a ) and an image of the lens front surface at 500 magnification showing no pigment particles ( b ) . 1dad : 1-day acuvue define . scanning electron microscopic ( sem ) images of cc . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the front surface ( fs ) of the lens ( a ) and an image of the lens front surface at 2,000 magnification showing visible pigment particles ( b ) . flc : freshlook one - day color . scanning electron microscopic ( sem ) images of nat . cross - sectional view at 2,000 magnification showing pigment on the front surface ( fs ) of the lens ( a ) and an image of the lens front surface at 2,000 magnification showing visible pigment particles ( b ) . the results of the afm analysis indicated that 1dad lenses had a statistically significantly lower rms value in the pigmented area than cc , dm2 , eco , tc , flc and nat contact lenses . table 3 displays the measures for each lens type by curve / print aspect combinations . table 4 and figure 8 summarise the statistical findings and graphically show the mean roughness values for each lens type in the different lens areas . figure 9 shows representative afm images of the pigmented areas for each lens type . atomic force microscopic average and range sd ( and range ) for root mean square ( rms ) and peak - to - peak values for the front and back surfaces , pigmented and non - pigmented areas 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . average root mean square ( rms ) roughness values determined by atomic force microscopy on the non - pigmented area of the front lens surface ( non - pig fs ) and back lens surface ( non - pig bs ) as well as the pigmented area of the front ( pig fs ) and back lens surfaces ( pig bs ) . pigment location was determined by scanning electron microscopy ( np : no pigment on the lens surface , bs : back surface pigment , fs : front surface pigment ) . 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . atomic force microscopic images of pigmented areas for 1dad ( a ) , tc ( b ) , cc ( c ) , dm2 ( d ) , eco ( e ) , nat ( f ) , and flc ( g ) . 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . statistical analysis of atomic force microscopy root mean square ( rms ) values for lenses found to have surface pigment versus the lens with pigments enclosed within the lens matrix 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . p - values are all versus 1dad additionally , 1dad lenses had roughness values that were more consistent between the pigmented and non - pigmented areas compared to the other lens types measured . rms roughness values for 1dad ranged from three to 18 nm , while rms values for the other lens tested ranged from two to 188 nm . cc , dm2 , eco and tc lenses were found to have pigments on the back surface ( concave side ) of the contact lens , while the pigments for nat and flc were found on the front surface ( convex side ) of the contact lens . the pigment particles were visible on the lens surface in top - down / bottom - up views , as well as in cross - sectional view appearing to extend from an average depth of 1.3 m to 3.5 m up to and including the lens surface for a depth range of 0.0 to 4.3m . the pigment particles for the 1dad were buried below the front surface of the lens at an average depth of 8.1 m with a range of 7.6 to 9.1 m ( table 2 ) . depth range for pigments from the lens surface when imaged by scanning electron microscope ( sem ) for each lens type 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable figures 1 7 show the cross - sectional depths at 2,000 magnification for each of the lens types . the images show no pigment particles on the top of the 1dad lenses , while a particle layer is on the surface of all other lenses tested . these results were used to determine the statistical comparisons to be completed for the afm findings ( surface pigmented lenses versus non - surface pigmented lenses ) . cross - sectional view at 2,000 magnification showing pigment enclosed within the lens matrix ( lm ) below the front lens surface ( a ) and an image of the lens front surface at 500 magnification showing no pigment particles ( b ) . 1dad : 1-day acuvue define . scanning electron microscopic ( sem ) images of cc . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the front surface ( fs ) of the lens ( a ) and an image of the lens front surface at 2,000 magnification showing visible pigment particles ( b ) . flc : freshlook one - day color . scanning electron microscopic ( sem ) images of nat . cross - sectional view at 2,000 magnification showing pigment on the front surface ( fs ) of the lens ( a ) and an image of the lens front surface at 2,000 magnification showing visible pigment particles ( b ) . the results of the afm analysis indicated that 1dad lenses had a statistically significantly lower rms value in the pigmented area than cc , dm2 , eco , tc , flc and nat contact lenses . table 3 displays the measures for each lens type by curve / print aspect combinations . table 4 and figure 8 summarise the statistical findings and graphically show the mean roughness values for each lens type in the different lens areas . figure 9 shows representative afm images of the pigmented areas for each lens type . atomic force microscopic average and range sd ( and range ) for root mean square ( rms ) and peak - to - peak values for the front and back surfaces , pigmented and non - pigmented areas 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . average root mean square ( rms ) roughness values determined by atomic force microscopy on the non - pigmented area of the front lens surface ( non - pig fs ) and back lens surface ( non - pig bs ) as well as the pigmented area of the front ( pig fs ) and back lens surfaces ( pig bs ) . pigment location was determined by scanning electron microscopy ( np : no pigment on the lens surface , bs : back surface pigment , fs : front surface pigment ) . 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . atomic force microscopic images of pigmented areas for 1dad ( a ) , tc ( b ) , cc ( c ) , dm2 ( d ) , eco ( e ) , nat ( f ) , and flc ( g ) . 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . statistical analysis of atomic force microscopy root mean square ( rms ) values for lenses found to have surface pigment versus the lens with pigments enclosed within the lens matrix 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . p - values are all versus 1dad additionally , 1dad lenses had roughness values that were more consistent between the pigmented and non - pigmented areas compared to the other lens types measured . rms roughness values for 1dad ranged from three to 18 nm , while rms values for the other lens tested ranged from two to 188 nm . the sem imaging results showed , both at the lens surface and throughout the depth of the lens , that the limbal ring pigment used for 1dad was enclosed within the lens matrix , and therefore would not be in direct contact with the ocular surface when worn . for the other lens types evaluated , each lens was found to have pigment at the surface level , some on the front surface and some on the back . the differences in pigment location are likely to be due to differences in the manufacturing processes . the testing methodology used for this analysis with afm measured the samples from similar locations , which resulted in a consistent comparison among the lenses tested . afm analysis showed 1dad lenses had lower roughness values ( that is , a smoother surface ) than the other limbal ring lenses tested within the pigmented area of the lens . additionally , 1dad had a more consistent roughness profile regardless of the curvature or pigmented side than any of the other limbal ring contact lenses tested . differences in surface roughness between the pigmented and non - pigmented areas are likely the result of pigment location ( due to the fact that this can be visualised at a low magnification ) but surface roughness of the non - pigmented areas can be related to manufacturing methods as well as material properties of the bulk material.22,23 although there are no thresholds of acceptable or non - acceptable rms roughness for contact lenses , values in the range of one to 11 nm for cast - moulded contact lenses can be considered normal and smooth . biomaterials research evaluating the effect of surface roughness and surface wettability on bacterial attachment to glass has suggested even nanoscale levels of roughness can impact bacterial adherence . mitik - dineva and colleagues23 found changes in surface wettability had no impact on bacterial attachment but the roughness of the material was found to play a role . although mitik - dineva and colleagues conducted their research using glass plates , later work published by giraldez and yebra - pimentel11 would suggest surface roughness is also a factor for bacterial adhesion with contact lenses . additional research is needed to determine to what effect pigment on the surface of a contact lens may have on product performance , including the adhesion of bacteria and the levels of bacteria that may be considered to be clinically relevant . it is important for eye - care providers to understand potential differences between limbal ring contact lenses , when determining what to prescribe for their patients , beyond the consumer - desired eye - enhancement effects . with the growing market and consumer interest in limbal ring lenses , pigment location and surface roughness although there has been some recent research reporting potential clinical implications with limbal ring contact lenses that have pigment on the surface of the lens , the clinical effects of pigment location of these lenses needs to be evaluated further .

introductiona relatively novel application for dermoscopy is its use in the monitoring of topical treatment response for non - melanoma skin cancer . basal cell carcinoma ( bcc ) is the most frequent type of skin cancer in humans . surgical excision is still considered the gold - standard of treatment . however , a number of topical therapies are now available for the treatment of different types of basal cell carcinoma.case reportthis case report exemplifies the usefulness of dermoscopy in the monitoring of residual disease after incomplete surgical excision and also in the monitoring of topical treatment response . imiquimod 5% cream acts as a topical immune response modifier promoting a th-1 immune response enhancing the removal of neoplastic cells and has proven to reduce deregulated hedgehog ( hh)/gli signal strength independent of toll - like receptor signaling , which makes it a valuable adjuvant topical therapy for the treatment of basal cell carcinoma.conclusionimiquimod 5% cream is a valuable adjuvant therapy for the treatment of incompletely excised bcc . this case report adds further evidence to the usefulness of dermoscopy in the assessment and monitoring of treatment outcome.electronic supplementary materialthe online version of this article ( doi:10.1007/s13555 - 015 - 0088-z ) contains supplementary material , which is available to authorized users . backgroundlimbal ring ( also known as circle ) contact lenses are becoming increasingly popular , especially in asian markets because of their eye - enhancing effects . the pigment particles that give the eye - enhancing effects of these lenses can be found on the front or back surface of the contact lens or enclosed within the lens matrix . the purpose of this research was to evaluate the pigment location and surface roughness of seven types of circle contact lenses.methodsscanning electron microscopic ( sem ) analysis was performed using a variable pressure hitachi s3400n instrument to discern the placement of lens pigments . atomic force microscopy ( dimension icon afm from bruker nano ) was used to determine the surface roughness of the pigmented regions of the contact lenses . atomic force microscopic analysis was performed in fluid phase under contact mode using a sharp nitride lever probe ( snl-10 ) with a spring constant of 0.06 n / m . root mean square ( rms ) roughness values were analysed using a generalised linear mixed model with a log - normal distribution . least square means and their corresponding 95% confidence intervals were estimated for each brand , location and pigment combination.resultssem cross - sectional images at 500 and 2,000 magnification showed pigment on the surface of six of the seven lens types tested . the mean depth of pigment for 1-day acuvue define ( 1dad ) lenses was 8.1 m below the surface of the lens , while the remaining lens types tested had pigment particles on the front or back surface . results of the atomic force microscopic analysis indicated that 1dad lenses had significantly lower root mean square roughness values in the pigmented area of the lens than the other lens types tested.conclusionssem and afm analysis revealed pigment on the surface of the lens for all types tested with the exception of 1dad . further research is required to determine if the difference in pigment location influences on - eye performance .

patient informed consent was not required for this retrospective analysis , though radiological examination - related informed consents were obtained from all patients before pet / ct and ct were performed . the inclusion criteria for this study were as follows : 1 ) pathologically confirmed malignant tumor in the periorbital region - we defined the periorbital region as the upper and lower eyelid and their appendages , in addition to the lacrimal glands , 2 ) both pet / ct and ct performed in each patient , with the interval between the two studies being less than one month . based on these criteria , 15 cases of periorbital malignant tumors were retrieved from the samsung medical center orbital cancer data registry for the period spanning 1996 to 2007 . the eyelid was the most commonly involved primary tumor site ( 11 cases , 73.3% ) ; other sites of involvement included the conjunctiva and lacrimal gland . pathological diagnoses included six sebaceous carcinomas ( 40.4% ) , four malignant melanomas ( 26.6% ) , two adenocarcinomas ( 13.3% ) , two adenoid cystic carcinomas ( 13.3% ) , and one squamous cell carcinoma ( 6.8% ) . surgical treatment alone was performed in 11 cases ( 73.4% ) , surgery with adjuvant radiotherapy in two cases ( 13.3% ) , and radiotherapy alone in two cases ( 13.3% ) . the median follow - up duration was 17.9 months ( range 3 - 52 months ) . eleven patients ( 73.4% ) have survived without evidence of disease , whereas two patients are still alive with disease , and two patients have died of disease . a total of 18 pet / ct and ct scans were performed in 15 cases . most of the scans were performed during the follow - up period ( 12 scans , 66.7% ) , and in some cases ( 6 scans , 33.3% ) , the scans were done as part of the initial diagnostic work - up . the reference standards for pet / ct and ct were surgical pathology ( n = 7 ) from dissected lymph node specimens and data from radiological follow - up ( n = 11 , duration 10 - 52 months , mean 20.5 months ) . radiological follow - up included two or more subsequent imaging studies ( ct , mri , or bone scans ) for suspected lesions during the follow - up period . ct scans ( lightspeed ultra or ultra 16 , ge , milwaukee , wi ) were performed using the following parameters : 160 mas , 120 kev , section width of 3.75 mm , and table feed of 8.75 mm per rotation . for contrast enhancement , 90 ml of an iodinated contrast agent ( ultravist 300 , schering , berlin ) was injected intravenously at 3 ml / sec using an automated injector . as for pet / ct scans , all patients fasted for at least six hours prior to the examinations , which were performed using a discovery ls pet / ct scanner ( ge healthcare , milwaukee , wi ) . whole body ct scanning was performed using a continuous spiral technique with an 8-slice helical ct with a gantry rotation speed of 0.8 sec . the ct scan data were collected using the following parameters : 80 mas , 140 kev , section width of 5 mm , and table feed of 5 mm per rotation . no intravenous or oral contrast agents were used . following the ct scan , and after the intravenous injection of 370 mbq f - fdg , an emission scan was performed from the thigh to the head at 5 minutes per frame , for a total of 45 minutes . the attenuation - corrected f - fdg pet images from the ct data were reconstructed with an ordered subset expectation maximization algorithm ( 28 subsets , 2 iterations ) . the images were displayed in a 128128 matrix ( pixel size = 4.294.29 mm , with a slice thickness of 4.25 mm ) . the separate ct and pet scan data were accurately co - registered using commercial software ( entegra , elgems , haifa , israel ) . the standardized uptake values ( suvs ) were acquired using the attenuation - corrected images , the amount of injected f - fdg , the body weight of each patient , and the cross - calibration factors between the f - fdg pet and the dose calibrator . for analysis , regional lymph nodes were divided into lymph node levels ( intra - parotid and peri - parotid lymph node groups , levels i , iia , iib , iii ) based on the ajcc staging manual ( 6th edition , 2002 ) . the peri - parotid lymph node group was defined to encompass the pre - auricular lymph nodes and the infra - parotid lymph nodes . pathological data were available in 23 dissected node levels , and radiological follow - up examination was used in 20 node levels . as a result , a total of 43 node levels were assessed with ct and pet / ct scans in the present study . a radiologist specializing in head and neck imaging reviewed the ct scans , and a nuclear medicine physician reviewed the pet / ct scans . the criteria for abnormal lymph nodes on ct included spherical or conglomerated shape , enlarged size ( > 1.5 cm at levels i and ii , > 1 cm at level iii ) , or enhancement pattern of contrast media . regarding the pet / ct scans , the nuclear medicine physician first reviewed the images to evaluate for any abnormal fdg uptake in the salivary glands and neck nodes by using the maximal uptake values with intensity higher than that of surrounding tissues . the interpretation was then revised based on the anatomical information provided by the combined pet / ct images . treatment planning was addressed in the multidisciplinary head and neck tumor conference along with the results of imaging studies . to compare the diagnostic accuracy of both imaging studies for regional metastasis , pathological data in the cases of surgery ( n = 7 ) or the follow - up results of radiological evaluation ( n = 11 ) were used as a reference standard . image findings were confirmed by radiological follow - up alone for eleven scans , with a mean follow - up duration of 20.5 months ( range 10 - 52 months ) . we compared the sensitivity ( sn ) , specificity ( sp ) , positive predictive value ( ppv ) , negative predictive value ( npv ) , and diagnostic accuracy ( da ) in predicting regional lymph node metastasis on level - by - level analysis and n staging . statistical differences between the imaging modalities were analyzed employing the mcnemar test , and 95% confidence levels were determined using wilson 's method . the impact of pet / ct on patient care was also assessed with regard to changes in the extent of surgery or in treatment planning . patient informed consent was not required for this retrospective analysis , though radiological examination - related informed consents were obtained from all patients before pet / ct and ct were performed . the inclusion criteria for this study were as follows : 1 ) pathologically confirmed malignant tumor in the periorbital region - we defined the periorbital region as the upper and lower eyelid and their appendages , in addition to the lacrimal glands , 2 ) both pet / ct and ct performed in each patient , with the interval between the two studies being less than one month . based on these criteria , 15 cases of periorbital malignant tumors were retrieved from the samsung medical center orbital cancer data registry for the period spanning 1996 to 2007 . the eyelid was the most commonly involved primary tumor site ( 11 cases , 73.3% ) ; other sites of involvement included the conjunctiva and lacrimal gland . pathological diagnoses included six sebaceous carcinomas ( 40.4% ) , four malignant melanomas ( 26.6% ) , two adenocarcinomas ( 13.3% ) , two adenoid cystic carcinomas ( 13.3% ) , and one squamous cell carcinoma ( 6.8% ) . surgical treatment alone was performed in 11 cases ( 73.4% ) , surgery with adjuvant radiotherapy in two cases ( 13.3% ) , and radiotherapy alone in two cases ( 13.3% ) . the median follow - up duration was 17.9 months ( range 3 - 52 months ) . eleven patients ( 73.4% ) have survived without evidence of disease , whereas two patients are still alive with disease , and two patients have died of disease . a total of 18 pet / ct and ct scans were performed in 15 cases . most of the scans were performed during the follow - up period ( 12 scans , 66.7% ) , and in some cases ( 6 scans , 33.3% ) , the scans were done as part of the initial diagnostic work - up . the reference standards for pet / ct and ct were surgical pathology ( n = 7 ) from dissected lymph node specimens and data from radiological follow - up ( n = 11 , duration 10 - 52 months , mean 20.5 months ) . radiological follow - up included two or more subsequent imaging studies ( ct , mri , or bone scans ) for suspected lesions during the follow - up period . ct scans ( lightspeed ultra or ultra 16 , ge , milwaukee , wi ) were performed using the following parameters : 160 mas , 120 kev , section width of 3.75 mm , and table feed of 8.75 mm per rotation . for contrast enhancement , 90 ml of an iodinated contrast agent ( ultravist 300 , schering , berlin ) was injected intravenously at 3 ml / sec using an automated injector . as for pet / ct scans , all patients fasted for at least six hours prior to the examinations , which were performed using a discovery ls pet / ct scanner ( ge healthcare , milwaukee , wi ) . whole body ct scanning was performed using a continuous spiral technique with an 8-slice helical ct with a gantry rotation speed of 0.8 sec . the ct scan data were collected using the following parameters : 80 mas , 140 kev , section width of 5 mm , and table feed of 5 mm per rotation . no intravenous or oral contrast agents were used . following the ct scan , and after the intravenous injection of 370 mbq f - fdg , an emission scan was performed from the thigh to the head at 5 minutes per frame , for a total of 45 minutes . the attenuation - corrected f - fdg pet images from the ct data were reconstructed with an ordered subset expectation maximization algorithm ( 28 subsets , 2 iterations ) . the images were displayed in a 128128 matrix ( pixel size = 4.294.29 mm , with a slice thickness of 4.25 mm ) . the separate ct and pet scan data were accurately co - registered using commercial software ( entegra , elgems , haifa , israel ) . the standardized uptake values ( suvs ) were acquired using the attenuation - corrected images , the amount of injected f - fdg , the body weight of each patient , and the cross - calibration factors between the f - fdg pet and the dose calibrator . for analysis , regional lymph nodes were divided into lymph node levels ( intra - parotid and peri - parotid lymph node groups , levels i , iia , iib , iii ) based on the ajcc staging manual ( 6th edition , 2002 ) . the peri - parotid lymph node group was defined to encompass the pre - auricular lymph nodes and the infra - parotid lymph nodes . pathological data were available in 23 dissected node levels , and radiological follow - up examination was used in 20 node levels . as a result , a total of 43 node levels were assessed with ct and pet / ct scans in the present study . a radiologist specializing in head and neck imaging reviewed the ct scans , and a nuclear medicine physician reviewed the pet / ct scans . the criteria for abnormal lymph nodes on ct included spherical or conglomerated shape , enlarged size ( > 1.5 cm at levels i and ii , > 1 cm at level iii ) , or enhancement pattern of contrast media . regarding the pet / ct scans , the nuclear medicine physician first reviewed the images to evaluate for any abnormal fdg uptake in the salivary glands and neck nodes by using the maximal uptake values with intensity higher than that of surrounding tissues . the interpretation was then revised based on the anatomical information provided by the combined pet / ct images . treatment planning was addressed in the multidisciplinary head and neck tumor conference along with the results of imaging studies . to compare the diagnostic accuracy of both imaging studies for regional metastasis , pathological data in the cases of surgery ( n = 7 ) or the follow - up results of radiological evaluation ( n = 11 ) were used as a reference standard . image findings were confirmed by radiological follow - up alone for eleven scans , with a mean follow - up duration of 20.5 months ( range 10 - 52 months ) . we compared the sensitivity ( sn ) , specificity ( sp ) , positive predictive value ( ppv ) , negative predictive value ( npv ) , and diagnostic accuracy ( da ) in predicting regional lymph node metastasis on level - by - level analysis and n staging . statistical differences between the imaging modalities were analyzed employing the mcnemar test , and 95% confidence levels were determined using wilson 's method . the impact of pet / ct on patient care was also assessed with regard to changes in the extent of surgery or in treatment planning . primary tumor evaluation information was only available in five of 18 scans ; the tumors were diagnosed correctly using pet / ct scans for the initial work - up in four cases and in one recurrent case . suvs in the primary tumors ranged from 2.9 to 7.5 ( median 4 ) . in our series , the maximum suv ranged from 2.2 to 8.1 ( mean 5.3 ) for lymph node metastases from sebaceous carcinoma , and from 2.0 to 38.7 from malignant melanoma . one patient with adenocarcinoma lymphatic metastasis showed fdg uptake in lymph nodes with maximum suv = 18.7 . on the lymph node level - by - level analysis , a total of 43 node levels were assessed with ct and pet / ct ( table 2 ) . there were 14 malignant node levels , with seven intra - parotid lymph nodes , three peri - parotid lymph nodes , one level i lymph node group , and three level iia lymph node groups . interestingly , metastasis to the cervical node levels ( i , ii , iii ) was always coincident with metastasis to peri- or intra - parotid nodes . ct had an sn of 57% , sp of 97% , ppv of 89% , npv of 82% , and da of 84% . for pet / ct , corresponding values were 100% , 97% , 93% , 100% , and 98% , respectively . of note , the difference in the sensitivity value between ct and pet / ct was statistically significant , with a p value of 0.03 . in the prediction of n staging according to the ajcc staging manual ( 6th edition , 2002 ) , pet / ct showed 100% accuracy ( 95% confidence interval : 82.4 - 100% ) , while ct showed 83% accuracy ( 95% confidence interval : 60.7 - 94.1% , p = 0.01 ) . the extent of the surgical field for regional lymph nodes was changed in the case of three pet / ct scans ( 3/18 , 16.6% ) . in two of the three cases , intra - parotid metastatic lymph nodes were detected on pet / ct , but not on ct . a 65-year - old male patient underwent eyelid and orbit exenteration secondary to biopsy - proven sebaceous carcinoma in the upper eyelid extending to the levator muscle and bulbar conjunctiva . after 16 months , a small node was detected in the ipsilateral parotid area during a follow - up visit . contrast - enhanced ct scan showed no abnormal mass lesion in the parotid area ( fig . pet / ct revealed a high glucose uptake lesion with an suv of 4.7 at the same site ; lymph node metastasis was strongly suspected . a parotidectomy was performed , and metastatic sebaceous carcinoma was diagnosed in an intra - parotid lymph node . another 67-year - old female patient was suspected of having recurrent sebaceous carcinoma in the parotid area . intra - parotid and peri - parotid metastatic lymph nodes were equally detected on ct and pet / ct , but the level ii lymph node was diagnosed as containing malignancy only on pet / ct . as a result , the surgical extent was expanded into the level ii area ; a metastatic lymph node was demonstrated pathologically . another 56-year - old male patient who had undergone orbital exenteration for sebaceous carcinoma in the upper eyelid was suspected of having regional recurrence in the ipsilateral parotid area ( fig . a 0.8 cm sized lymph node was detected just behind the angle of the mandible , but the contrast enhancement was not definite , and the lymph node maintained an oval shape , so a radiologist diagnosed it as an insignificant lymph node . however , pet / ct showed an asymmetrical lesion with an suv of 2.2 , suggesting metastasis . as a result , the extent of surgery was changed to include the above lymph node group , which proved to have metastasis on pathology . three patients in this study had distant metastases that were first detected by pet / ct . the pathology of the primary tumors included an adenocarcinoma in the upper eyelid , a melanoma in the conjunctiva , and an adenoid cystic carcinoma in the upper eyelid . pet / ct during the follow - up period detected distant metastases in the brain , pancreas , liver , stomach , or cervical spine , so treatment plans were changed to palliative care , including palliative chemotherapy or radiotherapy . in one case ( case no . 1 in table 1 ) , a malignant lymph node was suspected in the peri - parotid region on ct , contradictory to the pet / ct findings , which suggested a benign lymph node . surgical treatment was withheld until after confirmation using fine needle aspiration biopsy , and the patient still shows no evidence of disease in that area after 18 months of follow - up . as shown in table 3 , pet / ct affected treatment by correctly modifying the surgical extent or treatment decision in seven cases ( 38.8% ) . primary tumor evaluation information was only available in five of 18 scans ; the tumors were diagnosed correctly using pet / ct scans for the initial work - up in four cases and in one recurrent case . suvs in the primary tumors ranged from 2.9 to 7.5 ( median 4 ) . in our series , the maximum suv ranged from 2.2 to 8.1 ( mean 5.3 ) for lymph node metastases from sebaceous carcinoma , and from 2.0 to 38.7 from malignant melanoma . one patient with adenocarcinoma lymphatic metastasis showed fdg uptake in lymph nodes with maximum suv = 18.7 . on the lymph node level - by - level analysis , a total of 43 node levels were assessed with ct and pet / ct ( table 2 ) . there were 14 malignant node levels , with seven intra - parotid lymph nodes , three peri - parotid lymph nodes , one level i lymph node group , and three level iia lymph node groups . interestingly , metastasis to the cervical node levels ( i , ii , iii ) was always coincident with metastasis to peri- or intra - parotid nodes . ct had an sn of 57% , sp of 97% , ppv of 89% , npv of 82% , and da of 84% . for pet / ct , corresponding values were 100% , 97% , 93% , 100% , and 98% , respectively . of note , the difference in the sensitivity value between ct and pet / ct was statistically significant , with a p value of 0.03 . in the prediction of n staging according to the ajcc staging manual ( 6th edition , 2002 ) , pet / ct showed 100% accuracy ( 95% confidence interval : 82.4 - 100% ) , while ct showed 83% accuracy ( 95% confidence interval : 60.7 - 94.1% , p = 0.01 ) . the extent of the surgical field for regional lymph nodes was changed in the case of three pet / ct scans ( 3/18 , 16.6% ) . in two of the three cases , intra - parotid metastatic lymph nodes were detected on pet / ct , but not on ct . a 65-year - old male patient underwent eyelid and orbit exenteration secondary to biopsy - proven sebaceous carcinoma in the upper eyelid extending to the levator muscle and bulbar conjunctiva . after 16 months , a small node was detected in the ipsilateral parotid area during a follow - up visit . contrast - enhanced ct scan showed no abnormal mass lesion in the parotid area ( fig . pet / ct revealed a high glucose uptake lesion with an suv of 4.7 at the same site ; lymph node metastasis was strongly suspected . a parotidectomy was performed , and metastatic sebaceous carcinoma was diagnosed in an intra - parotid lymph node . another 67-year - old female patient was suspected of having recurrent sebaceous carcinoma in the parotid area . intra - parotid and peri - parotid metastatic lymph nodes were equally detected on ct and pet / ct , but the level ii lymph node was diagnosed as containing malignancy only on pet / ct . as a result , the surgical extent was expanded into the level ii area ; a metastatic lymph node was demonstrated pathologically . another 56-year - old male patient who had undergone orbital exenteration for sebaceous carcinoma in the upper eyelid was suspected of having regional recurrence in the ipsilateral parotid area ( fig . a 0.8 cm sized lymph node was detected just behind the angle of the mandible , but the contrast enhancement was not definite , and the lymph node maintained an oval shape , so a radiologist diagnosed it as an insignificant lymph node . however , pet / ct showed an asymmetrical lesion with an suv of 2.2 , suggesting metastasis . as a result , the extent of surgery was changed to include the above lymph node group , which proved to have metastasis on pathology . three patients in this study had distant metastases that were first detected by pet / ct . the pathology of the primary tumors included an adenocarcinoma in the upper eyelid , a melanoma in the conjunctiva , and an adenoid cystic carcinoma in the upper eyelid . pet / ct during the follow - up period detected distant metastases in the brain , pancreas , liver , stomach , or cervical spine , so treatment plans were changed to palliative care , including palliative chemotherapy or radiotherapy . in one case ( case no . 1 in table 1 ) , a malignant lymph node was suspected in the peri - parotid region on ct , contradictory to the pet / ct findings , which suggested a benign lymph node . surgical treatment was withheld until after confirmation using fine needle aspiration biopsy , and the patient still shows no evidence of disease in that area after 18 months of follow - up . as shown in table 3 , pet / ct affected treatment by correctly modifying the surgical extent or treatment decision in seven cases ( 38.8% ) . basal cell carcinoma and squamous cell carcinoma are the most common malignancies arising in the periorbital region , with malignant melanoma and sebaceous carcinoma occurring less frequently ( 2 ) . as in most solid tumors , regional metastases from periorbital malignancies spread through the lymphatic system , so careful evaluation of the regional lymph nodes is important . in this study , we included the three conjunctival melanomas for analysis , because they showed frequent metastasis to regional lymph nodes and distant sites , similar to other periorbital cancers . in a previous case study ( 6 ) , our group observed no direct tumor drainage to the submandibular lymph nodes - even from primary tumors located on the medial portion of the eyelid - without initial metastasis to the lymph nodes around the parotid gland ( first echelon lymph node ) ; this is concordant with the results of the present study . ct , with its high sensitivity ( 93% ) ( 17 ) , has been the mainstay imaging modality for diagnosing periorbital malignancies . however , the sensitivity of ct in the present study was as low as 57% . this is attributable to false negative lesions at peri - parotid and intra - parotid sites . some of these lesions were interpreted as inflammatory hyperplasia or infected cysts due to the absence of distinct features of malignant parotid tumors , such as lobular or irregular contour of the mass or ill - defined tumor margin . according to recent reports ( 18 , 19 ) , the higher sensitivity and diagnostic accuracy of pet / ct endows it with many advantages over ct alone in managing malignancies around the salivary gland , especially high - grade types . the present study also showed that pet / ct was effective in detecting lymphatic spread to the parotid region and cervical nodes in the setting of periorbital malignancies . among the 15 subjects enrolled in this study , seven had true regional lymph node metastasis ( 46.7% ) . the duration between the treatment of the primary tumor and the detection of regional recurrence ranged from 11 months to 240 months . on standard follow - up , the metastatic sites were diverse , including brain , cervical spine , pancreas , and lung . distant metastasis was detected in the cervical spine in one patient using pet / ct as an initial evaluation , and palliative treatment instead of curative surgery was implemented as a result . the higher sensitivity of pet / ct vindicated it as a useful screening method in the evaluation of regional lymph node metastasis , particularly in the follow - up setting . pet / ct had a higher npv ( 100% ) than did ct alone ( 82.4% ) , although this difference was not statistically significant . prediction of n stage was also more reliable with pet / ct than with ct alone . therefore , pet / ct can provide more accurate information about prognosis through revision of n staging of periorbital malignancies . in practice , how much diagnostic methods affect treatment decision - making is of great concern for clinicians . in this study , pet / ct had a positive impact on patient care by correctly modifying the treatment plan in approximately 40% of the patients . errors in interpretation of abnormal lymph node fdg uptake may be instigated in cases of larygopharyngeal inflammation . first , asymmetrical abnormal lymph node uptake strongly suggests metastasis , because inflammation frequently causes bilateral fdg uptake . malignant tumors in the periorbital area have lymph nodes around the parotid area as the first - echelon nodal group ( 6 ) . thus , without abnormal uptake in the lymph nodes around the parotid area , the increase of fdg uptake in the upper cervical lymph nodes , which commonly occurs in laryngo - pharyngitis , may be demarcated from the lymph node metastasis of periorbital malignancy . in addition , an suv of 2.0 in lymph nodes can be used as a cut - off value in determining the presence of metastasis , based on our results . first , this was a retrospective analysis with a small number of cases . prospective study with a larger number of cases is needed to fully assess the role of pet / ct in the management of lymph node metastasis in the setting of periorbital malignancies . second , heterogeneity of the neoplasms in the present study prompted the question of the radiographic equivalence of different tumors on pet / ct . characteristics of fdg uptake may vary among different pathologies , and it would be premature to claim that pet / ct is equally effective in all periorbital malignancies . third , our data does not answer the question of when to perform pet / ct in the setting of periorbital malignancies , though our results showed that pet / ct provided more accurate information about nodal status when lymphatic metastases were suspected . nevertheless , this study showed that pet / ct could provide more accurate diagnostic information regarding lymph node status . furthermore , it was more reliable in predicting n staging in the setting of periorbital malignancies than was ct alone . extra - hepatic hepatoid carcinomas are a rare group of aggressive tumors with clinical and pathologic features that closely resemble hepatocellular carcinoma ( hcc ) . they may arise in many areas outside of the liver including lungs , bladder , kidneys , uterus and ovaries , but most commonly in the stomach ( young et al . hepatoid carcinoma of the ovary ( hco ) is reported mainly in post - menopausal women with unilateral or bilateral ovarian masses and elevated serum alpha - fetoprotein ( afp ) . microscopically , these tumors demonstrate cells arranged predominantly in sheets and contain moderate to abundant amount of eosinophilic cytoplasm with pleomorphic nuclei ( lefkowitch , 1988 ) . these tumors must be distinguished from metastatic hcc and other afp - producing ovarian tumors including hepatoid yolk sac tumors ( hysts ) , sertoli leydig cell tumors , and dysgerminomas ( matsuta et al . , 1991 ) . a 73 year - old gravida 3 , para 2 was initially referred to her hematologist for anemia that was diagnosed during a prior hospital admission for legionella pneumonia . studies were consistent with a diagnosis of anemia of chronic disease and physical examination revealed a sizeable pelvic mass . the patient reported 60 lb weight loss and transient postmenopausal bleeding over the last year . ct of the abdomen and pelvis demonstrated a large pelvic mass with cystic and necrotic components measuring at least 18 cm by 17 cm with an additional 7 cm mass along the anterior peritoneal surface . marked bladder compression and left - sided hydronephrosis were also present . surgical exploration revealed a large , lobulated , necrotic mass originating from the left ovary ( fig . the mass was densely adherent to the uterus and anterior abdominal wall , and had eroded into a portion of small bowel . optimal cytoreductive surgery was performed including a total abdominal hysterectomy , bilateral salpingo - oophorectomy , and partial small bowel resection with reanastamosis . once a final histologic diagnosis was made , serum afp was obtained and was markedly elevated at 2396 ng / ml ( normal range 09 ) seven weeks after optimal cytoreduction . ct of the abdomen and pelvis at this time demonstrated new growth of a 4.7 cm nodular mass in the right upper quadrant separate from the liver and a 5.1 cm mass in the left side of the pelvis . afp values rapidly decreased with initiation of chemotherapy ( 273 27 6 4 3 ) . the patient completed six cycles of carboplatin ( auc of 6 , every 3 weeks ) and dose - dense paclitaxel ( 80 mg / m , every week ) with appropriate dose reductions secondary to development of severe anemia and thrombocytopenia . she is currently doing well with no evidence of recurrence 26 months after surgery . pathologic exam showed a 24 16.5 13 cm lobulated mass with tumor present on the outer surface . cut section showed a tan yellow , solid and cystic tumor which completely replaced the left ovary and fallopian tube . microscopically , the tumor showed extensive patchy tumor necrosis with nests and sheets of polygonal cells , most with clear cytoplasm and some showing granular eosinophilic cytoplasm ( fig . numerous pas positive and alpha - fetoprotein ( afp ) positive hyaline globules were scattered through the tumor ( fig . the tumor did not display the sinusoidal vascular pattern sometimes seen in both hco and hepatocellular carcinoma . immunohistochemical studies were diffusely strong positive for afp , keratin ae1/ae3 , arginase , and sall-4 , focally strong positive for heppar1 ; focally positive for cea and inhibin , and plap showed rare positive cells . negative staining was observed for calretinin , ema , oct4 , ck 7 , ck 20 , cd 30 , cd99 , and cd117 . primary hepatoid carcinoma of the ovary is a rare tumor that now has been reported in the literature thirty - two times since it was initially characterized in 1987 by ishikura and scully . the age range for patients is 35 to 78 years old with an average age of 56 . the tumor was bilateral in seven ( 22% ) of the reported cases and more than 75% of patients presented with stage iii or iv disease . rapid progression is also characteristic with a one and a two year survival rate of 83% and 53% respectively . the patient presented here has one of the largest reported hco tumors to date measuring 25 cm in its greatest dimension ( table 1 ) . sall-4 is regarded as a marker for germ cell tumors and is negative in hcc , but has been found in hepatoid carcinomas from other sites ( ushiku et al . , 2010 ) . keratins ae1/ae3 were positive in this case , consistent with an epithelial origin for the tumor . however , ck 7 and ck 20 , keratins often found in ovarian surface epithelial tumors , were negative . hcc is also usually negative for ck7 and ck20 , which are sometimes used together to help identify carcinomas of unknown primary . previous reported hco cases varied when tested for ck7 and ck 20 : 4 were ck7 positive , ck20 negative ; three were ck7 negative , ck20 negative ; and one was ck7 negative , ck20 positive ( table 2 ) . this tumor 's unique pathologic appearance must be distinguished namely from hysts and metastatic hcc . hysts tend to occur in a younger age group ( average age of 22 years ) , exhibit gonadal dysgenesis , and possess cellular uniformity with a lack of the giant bizzare cells with abundant cytoplasm ( trivedi et al . , 1998 ) . hcc must be excluded clinically and radiographically , as there is not yet any consistent pattern of ancillary lab studies to effectively rule it out . additional studies will be required to see if sall-4 will be a useful differential marker for hcc versus hco . ishikura and scully initially believed the tumor variant to be of surface epithelial origin and this view is supported by four reported cases of combined hcos admixed with surface epithelial carcinomas- two serous , one mucinous , one endometrioid ( ishikura and scully , 1987 , scurry et al . . additional support for this relationship is offered by the fact that hcos and surface epithelial tumors tend to occur in the same age group . an afp - producing serous papillary carcinoma in a 74 year - old woman has also been reported , although no hepatoid differentiation was present . cancer antigen 125 ( ca 125 ) is a non - specific marker that is usually seen in the serum and tumor tissue of patients with serous and endometrioid carcinomas yet serum ca 125 was elevated in 17 of 20 ( 85% ) cases and staining was positive in 5 of 11 ( 45% ) of the reported hco cases . furthermore , the abdominal cavity implants were serous papillary carcinoma following chemoradiation in case 1 of ishikura and scully 's case series of hcos ( ishikura and scully , 1987 , scurry et al . , 1996 ) . the embryologic connection of the yolk sac to the primitive gut is the origin of the hepatobiliary primordium so it seems possible to have hepatoid cells in yolk sac tumors . however , it is counterintuitive to accept that surface coelomic epithelium behaves like liver when there is no obvious embryologic connection . an unlikely explanation would be surface coelomic tumors whose differentiation regressed back to germ cell origins that were then redirected toward hepatoid features ( lefkowitch , 1988 ) . only one case report to date has documented a hco admixed with a sex cord stromal tumor of sertoli - type ( d'antonio et al . , 2010 ) . the best approach to treatment of this aggressive variant most regimens reported in the literature are platinum and taxane based therapy similar to those recommended for common epithelial ovarian tumors . the patient we presented in this case responded very well to carboplatin and dose - dense paclitaxel . of the 6 patients that were treated with carboplatin and paclitaxel , 5 reported survival outcomes and none were deceased at time of case publication : 100% 1 year survival rate ( range 1328 months ) . in one report , second - line treatment with sorafenib was initiated given its success as a first - line medication in the treatment of hcc , which pathologically resembles hco . however , the tyrosine kinase inhibitor proved ineffective as afp levels increased and a new tumor metastasis was noted in the liver prior to discontinuation of the medication ( pandey and truica , 2011 ) . a few cases have included intraperitoneal chemotherapy and radiation as well , but there is insufficient data to effectively compare and recommend treatment options . neither the histogenesis nor a consistent immunohistochemical profile of hco has been established , however afp staining is consistently positive with only one reported exception . both afp and ca125 appear to have prognostic value and can be used to follow response to treatment and screen for recurrence . treatment regimens have varied , but optimal cytoreductive surgery followed by a platinum and taxane based chemotherapy regimen has demonstrated outcomes similar to that seen with other ovarian carcinomas .

objectivethe aim of this study was to assess the clinical role of 18f - fdg pet / ct for the evaluation of lymph node metastasis in periorbital malignancies , compared with ct alone.materials and methodswe analyzed eighteen pet / ct and ct scans in 15 patients with biopsy - proven periorbital malignancies . we compared the diagnostic capabilities of pet / ct and ct with regard to nodal metastasis by level - by - level analysis and by n staging prediction . the reference standards were surgical pathology ( n = 7 ) from dissected lymph node specimens and the results from radiological follow - up ( n = 11 , mean 20.5 months ; range 10 - 52 months ) . moreover , any changes in patient care as prompted by pet / ct were recorded and compared with treatment planning for ct alone.resultspet/ct had a sensitivity of 100% , while ct had a sensitivity of 57% ( p = 0.03 ) for nodal metastasis by level - by - level analysis . pet / ct had a specificity of 97% , positive predictive value of 93% , negative predictive value of 100% , and diagnostic accuracy of 98% , while the ct values for these same parameters were 97% , 89% , 82% , and 84% , respectively . pet / ct correctly predicted n staging with an accuracy of 100% , while ct was only 83% accurate ( p = 0.01 ) . regarding the impact on patient care , the extent of surgery for regional lymph nodes and the treatment decision were modified by pet / ct in 39% of patients.conclusionpet/ct could provide useful information in the management of regional lymph node metastases in patients with periorbital malignancies . primary hepatoid carcinoma of the ovary ( hco ) is a rare aggressive tumor that typically presents at an advanced stage in postmenopausal women with unilateral or bilateral ovarian masses and elevated afp and ca125 . we report a case of hco in a 73 year - old woman who presented with abdominal distention , weight loss , and a large lower abdominal mass . postoperative serum afp was markedly elevated and trended down with initiation of chemotherapy . review of the literature revealed thirty - two reported cases with no consensus on histogenesis or consistent immunohistochemical profile other than positive afp staining in all but one case . although the optimal treatment has not yet been determined , tumor debulking surgery followed by a platinum and taxane based chemotherapy regimen has shown promise . both serum afp and ca125 appear to have prognostic value and can be used to follow response to treatment and screen for recurrence .

langerhans cell histiocytosis ( lch ) is a rare neoplastic disease of antigen - presenting cells , with an incidence rate of 4.05.4/1 million individuals . the most frequent sites of occurrence are skin , bone , central nervous system ( cns ) . computed tomography ( ct ) , magnetic resonance imaging ( mri ) , ultrasonic scan ( uss ) are mainly for the individual part , while multiple systems involvement need the whole body imaging . we present an unusual case of lch of the primary thyroid gland , which was confirmed by fine needle aspiration cytology . in addition to the thyroid gland , 18f - fluorodeoxyglucose ( 18f - fdg ) positron emission tomography ( pet)/ct discovered multiple organs involvement such as pituitary stalk , maxillofacial skin lesion , lungs , and skull . a 35-year - old man complained with painless progressive asymmetric enlargement of the bilateral thyroid lobes for a year . in the recent 3-month no hereditary syndromes , family history of cancer , and personal history were disclosed . there was no additional past medical or surgical history . blood tests revealed a normal full blood count , electrolytes , urea , creatinine , calcium , and phosphate levels . thyroid function tests reported a thyroid stimulating hormone level of 0.97 ( normal range 0.34.6 uiu / ml ) , free t4 of 21.78 ( normal range 6.624.8 pmol / l ) , thyroglobulin antibodies of 53.20 ( normal range < 30 u / ml ) , thyroid peroxidase antibody of 64.00 ( normal range 0.1610 iu / ml ) . in addition , serum calcitonin was recorded at < 1.0 ( normal range < 11.8 ng / l ) and urine catecholamine levels were within normal range . water pressure test revealed explicitly for central diabetes insipidus , and desmopressin acetate was given for 1-month . uss of the neck , conducted at the referring hospital , reported an enlarged ( 3.1 cm and 2.8 cm in diameter ) bilateral thyroid lobes [ figure 1 ] . subsequently , a needle biopsy from the thyroid mass revealed features of langerhans cell with the tumor cells staining positive for cd1a , s-100 , negative for cytokeratin [ figure 2 ] . 18fdg pet / ct was performed for further evaluation of lch in the whole body [ figure 4 ] . the maximum projection image of the pet revealed increased uptake in thyroid gland ( standardized uptake value [ suvmax ] = 9.4 ) [ figure 4a ] , pituitary stalk ( suvmax = 4.9 ) [ figure 4b ] , and maxillofacial skin lesion ( suvmax = 4.9 ) [ figure 4c ] , which corresponded well to bilateral thyroid nodule , the thicking of pituitary stalk , and skin lesion on the ct . however , ct showed the left lung nodule [ figure 4d ] and the skull destruction [ figure 4e ] without 18fdg uptake . ( a ) an ultrasonic scan ( uss ) of the neck , conducted at the referring hospital , reported multiple keyed acoustic area , basic clear boundary in both thyroid lobes [ b : 3.1 , and a : 2.8 cm in diameter ] . ( b ) an uss of the neck , conducted at the referring hospital , reported multiple keyed acoustic area , basic clear boundary in both thyroid lobes [ b : 3.1 and a : 2.8 cm in diameter ] ( a ) h and e staining [ a : h and e 100 ; b : h and e 400 ] and immunostaining ( cd1a , langerin and s-100 ) of lesions from the thyroid gland lesions . the lesions from the thyroid gland exhibited positive expression patterns for cd1a [ c : 100 ] , langerin [ d : 100 ] and s-100 [ e : 100 ] , and negative for cytokeratin ( ck ) [ f : 100 ] . ( b ) h and e staining [ a : h and e 100 ; b : h and e 400 ] and immunostaining ( cd1a , langerin and s-100 ) of lesions from the thyroid gland lesions . the lesions from the thyroid gland exhibited positive expression patterns for cd1a [ c : 100 ] , langerin [ d : 100 ] and s-100 [ e : 100 ] , and negative for ck [ f : 100 ] . ( c ) h and e staining [ a : h and e 100 ; b : h and e 400 ] and immunostaining ( cd1a , langerin and s-100 ) of lesions from the thyroid gland lesions . the lesions from the thyroid gland exhibited positive expression patterns for cd1a [ c : 100 ] , langerin [ d : 100 ] and s-100 [ e : 100 ] , and negative for ck [ f : 100 ] . ( d ) h and e staining [ a : h and e 100 ; b : h and e 400 ] and immunostaining ( cd1a , langerin and s-100 ) of lesions from the thyroid gland lesions . the lesions from the thyroid gland exhibited positive expression patterns for cd1a [ c : 100 ] , langerin [ d : 100 ] and s-100 [ e : 100 ] , and negative for ck [ f : 100 ] . ( e ) h and e staining [ a : h and e , 100 ; b : h and e , 400 ] and immunostaining ( cd1a , langerin and s-100 ) of lesions from the thyroid gland lesions . the lesions from the thyroid gland exhibited positive expression patterns for cd1a [ c : 100 ] , langerin [ d : 100 ] and s-100 [ e : 100 ] , and negative for ck [ f : 100 ] . ( f ) : h and e staining [ a : h and e 100 ; b : h and e 400 ] and immunostaining ( cd1a , langerin and s-100 ) of lesions from the thyroid gland lesions . the lesions from the thyroid gland exhibited positive expression patterns for cd1a [ c : 100 ] , langerin [ d : 100 ] and s-100 [ e : 100 ] , and negative for ck [ f : 100 ] ( a ) magnetic resonance imaging ( mri ) sections of the head revealing pituitary stalk enlargement . ( b ) mri sections of head revealing pituitary stalk enlargement ( a ) 18f - fluorodeoxyglucose positron emission tomography / computed tomography ( 18fdg pet / ct ) revealed increased uptake in the thyroid gland and maxillofacial skin , in addition to the left lung and skull . ( b ) 18fdg pet / ct revealed increased uptake in the thyroid gland and maxillofacial skin , in addition to the left lung and skull . ( c ) 18fdg pet / ct revealed increased uptake in the thyroid gland and maxillofacial skin , in addition to the left lung and skull . ( d ) 18fdg pet / ct revealed increased uptake in the thyroid gland and maxillofacial skin , in addition to the left lung and skull . ( e ) 18fdg pet / ct revealed increased uptake in the thyroid gland and maxillofacial skin , in addition to the left lung and skull langerhans cell histiocytosis is a type of histiocytic disorders , which was first reported in 1893 . the age of onset of lch from infant to adult and it frequently occur in childhood . final diagnosis of lch is made by histopathology and immunohistochemistry , and there are no specificities only depending on the clinical features or radiologic findings of lch . the radiologic imaging may provide some characteristics , such as punched - out lesion or beveled edge in skull , symmetric flattening of vertebral body , lytic lesion with sclerotic rim and surrounding areas of sclerosis more common in iliac bone , diaphyseal or metaphyseal of long bones involved in children . otherwise , approximately 70% of patients will also show thickening of the pituitary stalk on mr images . the mechanism of fdg uptake by malignant tissue or metastatic lesions is due to the enhanced rate of glucose utilization by neoplastic cells . lch as a type of histiocytic disorders , most of the lesions display a high level of fdg uptake . the lesions of lch can range from a single system involvement to a multisystem disease . it may present as a neoplasm , skin rash , lytic bone lesion , emphysematous bullae , interstitial lung disease or present with multiple affected organ systems within the human body . the most common clinical cns manifestation of lch is diabetes . in this case , the patient underwent a whole - body pet / ct , which was positive for suspicious lesions , pet revealed increased fdg uptake not only in thyroid nodule , but also in the pituitary stalk and maxillofacial skin . in the region of these areas , the intense fdg activity on pet corresponded well to ct and the fusion images . we also found that the left lung nodule has slighter metabolic ( suvmax 0.5 ) and the skull destruction even nonmetabolic in pet . it has been reported that pet may showed false - negative in some small bone lesions , such as skull and cerebrum involvement . hence , it was suggested that combined multiple analysis methods would improve sensitivity and specificity of the whole body lch . positron emission tomography / computed tomography is a useful tool to rule out the presence of other organ infiltration . as the intent by our case , it also provides a reference basis of classification , staging , treatment plan , and evaluation therapy effect . fdg pet / ct not only provides the characteristics of lesions in ct scan , but also finding the lesions activity fdg uptake . previous reports of lch in fdg pet / ct imaging mostly shows bone , lung , lymph node or skin invasion . few cases of the primary thyroid lch showed in fdg pet / ct have been reported . in the patient of this case , the thyroid involved lch was the first symptoms , and clinical considered as thyroid cancer . whole - body pet / ct was performed to evaluate the involvement of disease and make an individualized treatment plan after the biopsy pathology . fdg pet / ct showed bilateral involvement of multiple low - density nodules , the boundary of nodules are clear and some breakthrough thyroid capsule . fdg uptake was significantly increased in the nodules ; which were mostly higher than primary thyroid cancer in addition to low - differentiated cancer and medullary carcinoma . in ct , these nodules had rare calcification and the density mostly lower than primary thyroid cancer . however , to confirm all these characteristic performances , needs more patients data accumulation . in summary , we recommend the use of pet / ct as a meaningful noninvasive tool to evaluation scope of lch lesions involving . major depressive disorder ( mdd ) is the most common mood disorder with a significant effect on the progression of medical conditions.1 factors accompanying depression , such as patients ' failure to look after their health , inability to adapt to their environment , social dissociation , chronic stresses of life , cigarette smoking , reduced physical activity , inappropriate nutrition and poor compliance with medical advice , make depression one of the risk factors of noncommunicable diseases.2 in addition to the effect of depression on lifestyle , the direct effect of depression on metabolic factors has been shown in many studies.3 recently , a relationship has been observed between depression and serum levels of lipoproteins and apolipoproteins ( apo ) , which are known risk factors of obesity and cardiovascular diseases.4 one theory of this relationship suggests a disturbance in function of the serotonergic system . in addition , metabolic changes in patients with mdd are due to genetic changes in the coding of serum lipoproteins.5 other theories describe changes in interleukin 2 , number of total tcells , melatonin and other cytokines in depressed patients.6 - 8 despite these studies , controversy exists about the relationship between depression and the lipid profile . studies have shown different results for the level of apo a one of the highdensity lipoprotein cholesterol ( hdlc ) subgroups , and of apo b the major protein of lowdensity lipoprotein cholesterol ( ldlc ) , in patients with mdd.4,9,10 sevincok and sarandol showed that the serum level of apo a in depressed patients was lower than that in control group.4,9 another study showed no significant difference in the serum levels of apolipoproteins between depressed patients and the control group.11 the lack of evidence and controversial results of previous studies led us design this study to compare the serum levels of apolipoproteins in depressed patients and normal individuals . a population of 153 patients with mdd ( 63 women , 90 men , aged 2147 years ) in 2007 were included in this case control study . all the patients were diagnosed with mdd according to the structured clinical interview ( scidi ) for diagnostic and statistical manual of mental disorders , fourth edition ( dsmiv ) . relatives of hospitalized patients and hospital university staff comprised the control group ( 78 women , 69 men , aged 1847 years ) . patients with an axis i or ii disorder in addition to their depression , patients with mdd with psychotic features , bipolar disorder , cyclothymia , dysthymia , anxiety disorder and patients at significant risk of suicide were excluded from the study via the structured clinical interview for dsmiv . exclusion criteria for both case and control groups included the presence of organic diseases such as hypertension , diabetes , cardiovascular , adrenal , hepatic and thyroid diseases documented by physical examination and laboratory tests ; history of antilipid and blocker consumption ; and menopause in women . laboratory tests , including complete blood count , serum electrolyte assay , liver function tests , thyroid function tests , urine analyses and electrocardiography , were performed for all participants to screen for major health problems . in addition , after explanation of the study , informed consent was obtained from all participants . the study protocol was approved by the ethical committee of the isfahan university of medical sciences . all participants completed a selfadministered questionnaire to determine demographic characteristics such as age , gender , socioeconomic status ( occupation , marital status and educational level ) , smoking status , drug history , family history of depression , physical activity and diet . subjects were considered to be current smokers if they reported smoking at least one cigarette a day during the past year . anthropometric characteristics ( height and weight ) were measured with the subjects wearing thin clothes . body mass index ( bmi ) was calculated by dividing a participant 's weight by their height squared ( kg / m ) . regular physical activity was defined as exercise of at least 15 minutes ' duration at least twice each week.13 participants provided details of their dietary habits by completing a food frequency questionnaire ( ffq).14 this instrument was designed according to the whoffq , with some changes . the validity of this questionnaire was confirmed before its use by the medical education development center affiliated to isfahan university of medical sciences.15 the ffq was used to access the consumption of different food groups such as meat , oils , cereals , vegetables and fruits . eligible participants also completed a 17item hamilton rating scale for depression ( hamd ) , which is the most commonly used observerrated depressive symptom rating scale . the original scale has 21 items but , as hamilton has suggested , only the initial 17 items were scored in this study because the last 4 items rarely occur and deal only with aspects of illness . items with quantifiable severity were ranked on a scale of 04 and those measuring symptoms that are difficult to assess reliably were ranked on a scale of 02 . the range of the 17item scale was 050 , with 14 considered to be the cutoff point of this scale ; higher scores indicated more severe depression . according to hamd , patients were classified into 3 groups of mild mdd ( score 1518 ) , moderate mdd ( score 1922 ) and severe mdd ( score 23).16,17 a blood sample was taken after 1214 hours ' fasting through the antecubital vein . all the blood sampling procedures were performed in the central laboratory of the isfahan cardiovascular research centre . serum triglyceride ( tg ) , total cholesterol ( tc ) , hdlc , ldlc and apo a and b were measured for each patient . tc and tg levels were measured within 24 h by an enzymatic method using an elan 2000 autoanalyze . ldlc was calculated by the friedewald formula,18 but in cases with tg400 mg / dl , it was measured directly . apo a and b were measured by immunoturbidimetry using pars azmoon kits accredited by bioactiva diagnostica ( germany ) . continuous variables were expressed as mean sd and a ttest was used to compare the means between the two groups . qualitative variables were expressed as frequency and a test used to compare frequencies between the two groups . a pearson correlation test was used to evaluate the correlation of depression with apo a and apo b. to study the association of depression and apo a and b ( apo a and b , separately ) , multiple linear regression models were used . apo a and b were entered as dependent variables , and the group ( case vs. control ) , smoking status ( smokers vs. nonsmokers ) , bmi , tc , ldlc and hdlc were entered as independent variables . the enter approach was selected for regression model , and for each apo a and b a unique model was created . data were analyzed by spss version 15.0 ( spss inc , chicago , illinois , usa ) . patients with an axis i or ii disorder in addition to their depression , patients with mdd with psychotic features , bipolar disorder , cyclothymia , dysthymia , anxiety disorder and patients at significant risk of suicide were excluded from the study via the structured clinical interview for dsmiv . exclusion criteria for both case and control groups included the presence of organic diseases such as hypertension , diabetes , cardiovascular , adrenal , hepatic and thyroid diseases documented by physical examination and laboratory tests ; history of antilipid and blocker consumption ; and menopause in women . laboratory tests , including complete blood count , serum electrolyte assay , liver function tests , thyroid function tests , urine analyses and electrocardiography , were performed for all participants to screen for major health problems . in addition , after explanation of the study , informed consent was obtained from all participants . the study protocol was approved by the ethical committee of the isfahan university of medical sciences . all participants completed a selfadministered questionnaire to determine demographic characteristics such as age , gender , socioeconomic status ( occupation , marital status and educational level ) , smoking status , drug history , family history of depression , physical activity and diet . subjects were considered to be current smokers if they reported smoking at least one cigarette a day during the past year . anthropometric characteristics ( height and weight ) were measured with the subjects wearing thin clothes . body mass index ( bmi ) was calculated by dividing a participant 's weight by their height squared ( kg / m ) . regular physical activity was defined as exercise of at least 15 minutes ' duration at least twice each week.13 participants provided details of their dietary habits by completing a food frequency questionnaire ( ffq).14 this instrument was designed according to the whoffq , with some changes . the validity of this questionnaire was confirmed before its use by the medical education development center affiliated to isfahan university of medical sciences.15 the ffq was used to access the consumption of different food groups such as meat , oils , cereals , vegetables and fruits . eligible participants also completed a 17item hamilton rating scale for depression ( hamd ) , which is the most commonly used observerrated depressive symptom rating scale . the original scale has 21 items but , as hamilton has suggested , only the initial 17 items were scored in this study because the last 4 items rarely occur and deal only with aspects of illness . items with quantifiable severity were ranked on a scale of 04 and those measuring symptoms that are difficult to assess reliably were ranked on a scale of 02 . the range of the 17item scale was 050 , with 14 considered to be the cutoff point of this scale ; higher scores indicated more severe depression . according to hamd , patients were classified into 3 groups of mild mdd ( score 1518 ) , moderate mdd ( score 1922 ) and severe mdd ( score 23).16,17 a blood sample was taken after 1214 hours ' fasting through the antecubital vein . all the blood sampling procedures were performed in the central laboratory of the isfahan cardiovascular research centre . serum triglyceride ( tg ) , total cholesterol ( tc ) , hdlc , ldlc and apo a and b were measured for each patient . tc and tg levels were measured within 24 h by an enzymatic method using an elan 2000 autoanalyze . ldlc was calculated by the friedewald formula,18 but in cases with tg400 mg / dl , it was measured directly . apo a and b were measured by immunoturbidimetry using pars azmoon kits accredited by bioactiva diagnostica ( germany ) . continuous variables were expressed as mean sd and a ttest was used to compare the means between the two groups . qualitative variables were expressed as frequency and a test used to compare frequencies between the two groups . a pearson correlation test was used to evaluate the correlation of depression with apo a and apo b. to study the association of depression and apo a and b ( apo a and b , separately ) , multiple linear regression models were used . apo a and b were entered as dependent variables , and the group ( case vs. control ) , smoking status ( smokers vs. nonsmokers ) , bmi , tc , ldlc and hdlc were entered as independent variables . the enter approach was selected for regression model , and for each apo a and b a unique model was created . data were analyzed by spss version 15.0 ( spss inc , chicago , illinois , usa ) . the baseline variables sex , age , marital state and occupation were similar in case and control groups . the mean age of participants was 31.2110.41 years in the group with mdd vs. 32.008.21 years in the control group . depressed patients were significantly more likely to have a family history of depression and a past history of smoking ( p<0.05 ) . fiftyfour patients were mildly depressed ( 35.3% ) , 69 were moderately depressed ( 45.1% ) and 30 were severely depressed ( 19.6% ) . there were no statistically significant differences between the two groups in bmi and physical activity . consumption of different groups of foods , which was assessed by the ffq , showed no significant statistical difference between the two groups . concentrations of all serum lipids and apo a and b differed significantly between the two groups . tc , ldlc and apo b were higher , while hdlc and apo a were lower , in the case group than in controls ( table 1 ) . linear regression analysis showed that serum apo a levels were negatively ( p<0.01 ) and serum apo b levels were positively ( p<0.05 ) predicted by depression . serum tc levels predicted negatively ( p<0.05 ) and hdlc levels predicted positively ( p<0.01 ) the serum apo a levels . smoking status and bmi did not significantly predict the apo a and b levels ( table 2 ) . the correlation between depression severity according to hamd and serum levels of the apolipoproteins indicated an inverse relationship between depression severity and serum apo a levels ( r = 0.453 , p<0.01 ) and a direct relationship between depression severity and serum apo b levels ( r = 0.521 , p<0.05 ) . our findings showed a significant correlation between serum levels of apolipoproteins and depression so that the serum level of hdlc and apo a were lower , while ldlc and apo b ( atherogenic lipoproteins ) were higher , in patients with mdd than those in the control group . in addition , severity of depression correlated with an increment in serum apo b level and a decrement in serum apo a level . sarandol et al . investigated the oxidation of apo bcontaining lipoproteins and the serum paraoxonase and arylesterase activities in mdd . their case group included patients with mdd who had not received antidepressant drugs for at least 3 weeks . higher tc , hdlc , ldlc and apo b , and lower apo a , levels were found in the case group . the patients were treated with a standard dosage of antidepressant drugs for 6 weeks , which did not alter the serum levels of lipid profiles.4 however , there is some evidence that antidepressant agents may affect serum lipid profile levels.9 thus , we excluded patients who had taken antidepressant drugs during the past 6 months . our results , except those for hdlc , were in line with those of sarandol et al . association between serum hdlc and ldlc levels as predictors of coronary heart disease ( chd ) and mdd is one of the fields which has been investigated.19 few studies have shown an inverse association between them.2 a metaanalysis on the association of cholesterol and depression showed that tc and depression were inversely related.21 on the other hand , some studies , such as that of chen et al . , demonstrated lower hdlc and higher ldlc and tc levels in patients with mdd.22 similarly , nakao and yano showed significant direct association between hypercholesterolemia and patients with mdd in japanese men.23 since apo a is known to be a major fraction of hdlc and apo b is known to be a major fraction of ldlc , our results were in accordance with the results of these studies . depression is associated with chd that is , depressed patients are more prone to develop chd.24,25 because of this association , the relationship between depression and other risk factors of chd such as serum apolipoprotein levels is an important subject that should examined more closely . one hypothesis suggests that genetic factors associated with depression may contribute to a change in serum lipid levels.26 a second theory proposes that cholesterol may be an important factor in reducing the possibility of correction of metabolic defects . as a result of these defects , segmental cerebral hypoxia , which may associated with depression , could occur.27 altered lipid profile levels through changes in serotonergic systems might also lead to mdd.28 this study has some limitations . owing to the crosssectional design of the study , the cause and effect relationship and mechanisms of the association between depression and lipid profile could not be determined . also , some of our data were based on selfreported questionnaires , which are less reliable sources of information than direct measurement . also , as previously mentioned , although our case and control groups had no differences in socioeconomic status , some of the control group comprised university hospital staff . the results of the study demonstrated lower serum levels of apo a and hdlc and higher serum levels of apo b and ldlc in depressed patients than in the control group . thus in depressed patients , biochemical problems should be considered and evaluated together with psychological interventions . given the relationship between apolipoproteins and depression , checking the lipid profile as predicting factors of chd to prevent the appearance of extra cardiovascular risk factors seems necessary .

langerhans cell histiocytosis ( lch ) is a rare clonal proliferative disease , with an incidence rate of 4.05.4/1 million individuals . lch encompasses a spectrum of disorders with diverse clinical presentations ranging from a single organ to multiple organ involvement . lch rarely involves the thyroid gland . we presented a case with lch of thyroid gland . the patient had painless progressive neck enlargement and then diabetes insipidus . ultrasonic scan and magnetic resonance imaging scan revealed nodular goiter and pituitary stalk enlargement , respectively . histopathological analysis revealed features of histiocytoid cells . 18f - fluorodeoxyglucose ( 18f - fdg ) positron emission tomography / computed tomography ( pet / ct ) was performed in order to rule out the presence of whole body infiltration . 18f - fdg pet / ct also demonstrated increased uptake in the thickening pituitary stalk and maxillofacial skin lesion , in addition to the bilateral thyroid nodules , ct showed the left lung nodule and the skull destruction without 18f - fdg uptake . this report emphasizes the role of 18f - fdg pet / ct in multiple organs involvement of patients with lch . objective : to investigate the relation between major depressive disorder and metabolic risk factors of coronary heart disease.introduction:little evidence is available indicating a relationship between major depressive disorder and metabolic risk factors of coronary heart disease such as lipoprotein and apolipoprotein.methods:this case control study included 153 patients with major depressive disorder who fulfilled the criteria of the diagnostic and statistical manual of mental disorders , fourth edition ( dsmiv ) , and 147 healthy individuals . all participants completed a demographic questionnaire and hamilton rating scale for depression . anthropometric characteristics were recorded . blood samples were taken and total cholesterol , high and lowdensity lipoproteins and apolipoproteins a and b were measured . to analyze the data , ttest , 2 test , pearson correlation test and linear regression were applied.results:depression was a negative predictor of apolipoprotein a ( = 0.328 , p<0.01 ) and positive predictor of apolipoprotein b ( = 0.290 , p<0.05 ) . apolipoprotein a was inversely predicted by total cholesterol ( = 0.269 , p<0.05 ) and positively predicted by highdensity lipoprotein ( = 0.401 , p<0.01 ) . also , lowdensity lipoprotein was a predictor of apolipoprotein b ( = 0.340 , p<0.01 ) . the severity of depression was correlated with the increment in serum apolipoprotein b levels and the decrement in serum apolipoprotein a level.conclusion:in view of the relationship between apolipoproteins a and b and depression , it would seem that screening of these metabolic risk factors besides psychological interventions is necessary in depressed patients .

osteoporosis is defined as a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fractures associated with chronic pain , disability , and mortality 1 . osteoporosis is caused by a disturbance of the number or activity of osteoclasts , resulting in inappropriately high bone resorption , which exceeds the capacity of osteoblasts . bone strength is an integration of bone density and bone quality , and bone mineral density ( bmd ) accounts for approximately 70% of bone strength 1,2 . osteoporosis has become a significant public health problem throughout the world , and the identification of risk factors related to osteoporosis is important to predict and prevent this disease . estrogen maintains a balance between osteoclastic and osteoblastic activity , and bone remodeling increases when estrogen levels decline 3 . hormone levels are the main determinants of bone density ; however , other factors such as smoking , excessive alcohol consumption , lean body mass , low levels of physical activity , and the presence of other medical conditions , including chronic renal disease , hyperparathyroidism , hyperthyroidism , and diseases requiring systemic corticosteroid use , also increase the risk of osteoporosis 4 - 6 . a variety of neoplasms without bone metastasis are also known to be related to osteoporosis by producing circulating bone resorption stimulatory factors , leading to bone destruction and hypercalcemia 7 - 12 . in patients with gynecologic cancers , reduced spinal bmd has been reported in patients with cervical cancer 13,14 , but no significant differences were found in spinal or femoral bmd between patients with endometrial cancer and controls 15 . we hypothesized that hormone - dependent tumor , at least endometrial cancer , may preserve the bmd , contrary to cervical cancer . the aims of the present study were to compare the bone turn - over markers , bmd , and frequency of osteoporosis or osteopenia at the lumbar spine and femur between patients with cervical or endometrial cancer and controls . furthermore , we compared the bone turn - over markers and bmd base on their cancer stages . in this cross - sectional study , patients aged 45 - 57 years who first visited 3 university hospitals ( kosin university , wonkwang university , and inje university ) and were diagnosed with cervical or endometrial cancer without bone metastasis between january 2008 and december 2013 were included in the study . cervical cancer was diagnosed by papanicolaou smear and colposcopically directed biopsy , and endometrial cancer was initially diagnosed by dilatation and curettage of the uterus . technetium-99m - labeled diphosphonate bone scans or 18f - fluorodeoxyglucose positron emission tomography / computed tomography were performed on all cancer patients for confirmation of bone metastasis . all participants received dual - energy x - ray absorptiometry ( dxa ) at the time of diagnosis before any cancer treatment . study subjects who had not reached menopause or who received menopausal hormone therapy were excluded . postmenopausal women aged 48 - 59 years who visited the university hospitals as part of a group check - up for work and lacked specific health problems served as normal controls . all control women underwent a careful physical examination and a thorough review of medical history , and the subjects who had history of current treatment with drugs known to alter bone or calcium metabolism were excluded . finally , 218 patients with cervical cancer , 85 patients with endometrial cancer , and 259 healthy controls were enrolled in this study . bmd data of the lumbar spine and femur and laboratory data of bone turnover markers were collected for all participants . bmd was measured in grams per square centimeter at the first lumbar spine vertebrae ( l1),l2 , l3 , l4 and the femur , using dxa ( lunar radiation corp , madison , wi , usa ) . we used the t - score of total lumber spine , total hip , or femoral neck as single measurement for the diagnosis of osteoporosis . bmd values were categorized into three groups according to the criteria of the world health organization 16 and official positions 2013 of international society for clinical densitometry 17 as normal , osteopenic , or osteoporotic relative to the mean and standard deviation of young women . osteoporosis group was classified as women whose t - score of the total lumbar spine , total hip , or femur neck was -2.5 or less . normal group was composed with the women whose t - score of the total lumbar spine , proximal hip , and femur neck were over -1.5 . body mass index ( bmi ) was calculated by dividing bodyweight ( kg ) by the square of body height ( m ) . blood samples were collected from all participants in tubes without anticoagulants , and sera were obtained by centrifugation or determination of bone turn - over markers . all statistical analyses were performed using spss version 19.0 ( spssinc . , chicago , il , usa ) . for comparisons of demographic and anthropometric characteristics , serum and urine biochemical markers , and t -scores of basal bmd between patients with cervical cancer , those with endometrial cancer , and healthy controls , student 's t - test was performed . for comparison of these parameters in cancer patients categorized according to cancer stage , the student 's t - test was used . the frequencies of osteoporosis , osteopenia , and normal bmd according to basal bone mass were compared between the cancer groups and the healthy control group using the test . the analysis between relatively normal bmd and decreased bmd according to the existence of cancer was carried out with logistic regression test . in this cross - sectional study , patients aged 45 - 57 years who first visited 3 university hospitals ( kosin university , wonkwang university , and inje university ) and were diagnosed with cervical or endometrial cancer without bone metastasis between january 2008 and december 2013 were included in the study . cervical cancer was diagnosed by papanicolaou smear and colposcopically directed biopsy , and endometrial cancer was initially diagnosed by dilatation and curettage of the uterus . technetium-99m - labeled diphosphonate bone scans or 18f - fluorodeoxyglucose positron emission tomography / computed tomography were performed on all cancer patients for confirmation of bone metastasis . all participants received dual - energy x - ray absorptiometry ( dxa ) at the time of diagnosis before any cancer treatment . study subjects who had not reached menopause or who received menopausal hormone therapy were excluded . postmenopausal women aged 48 - 59 years who visited the university hospitals as part of a group check - up for work and lacked specific health problems served as normal controls . all control women underwent a careful physical examination and a thorough review of medical history , and the subjects who had history of current treatment with drugs known to alter bone or calcium metabolism were excluded . finally , 218 patients with cervical cancer , 85 patients with endometrial cancer , and 259 healthy controls were enrolled in this study . bmd data of the lumbar spine and femur and laboratory data of bone turnover markers were collected for all participants . bmd was measured in grams per square centimeter at the first lumbar spine vertebrae ( l1),l2 , l3 , l4 and the femur , using dxa ( lunar radiation corp , madison , wi , usa ) . we used the t - score of total lumber spine , total hip , or femoral neck as single measurement for the diagnosis of osteoporosis . bmd values were categorized into three groups according to the criteria of the world health organization 16 and official positions 2013 of international society for clinical densitometry 17 as normal , osteopenic , or osteoporotic relative to the mean and standard deviation of young women . osteoporosis group was classified as women whose t - score of the total lumbar spine , total hip , or femur neck was -2.5 or less . normal group was composed with the women whose t - score of the total lumbar spine , proximal hip , and femur neck were over -1.5 . body mass index ( bmi ) was calculated by dividing bodyweight ( kg ) by the square of body height ( m ) . blood samples were collected from all participants in tubes without anticoagulants , and sera were obtained by centrifugation or determination of bone turn - over markers . all statistical analyses were performed using spss version 19.0 ( spssinc . , chicago , il , usa ) . for comparisons of demographic and anthropometric characteristics , serum and urine biochemical markers , and t -scores of basal bmd between patients with cervical cancer , those with endometrial cancer , and healthy controls , student 's t - test was performed . for comparison of these parameters in cancer patients categorized according to cancer stage , the student 's t - test was used . the frequencies of osteoporosis , osteopenia , and normal bmd according to basal bone mass were compared between the cancer groups and the healthy control group using the test . the analysis between relatively normal bmd and decreased bmd according to the existence of cancer was carried out with logistic regression test . all patients who were diagnosed with cervical cancer underwent type i or ii hysterectomy and pelvic lymphadenectomy . the distribution of the international federation of gynecology and obstetrics ( figo ) stage in the cervical cancer patients was ib , 122 ( 55.96% ) and iia , 96 ( 44.04% ) . of these 218 patients , 178 had squamous cell carcinoma , 27 had adenocarcinoma , 9 had adenosquamous carcinoma , and four had other types of cancer . patients with endometrial cancer were initially diagnosed by dilatation and curettage of the uterus , and were pathologically proven after staging operations including pelvic lymph node ( ln ) dissection or para - aortic ln dissection . the distribution of surgical figo stage was ia , 67 ( 78.82% ) ; ib , 9 ( 10.59% ) ; iia , 8 ( 9.41% ) ; and iib , one ( 1.18% ) . of these patients , 68 had endometrioid adenocarcinoma , 12 had squamous cell carcinoma , and five had papillary serous adenocarcinoma . age , bmi , parity , and time since menopause did not differ significantly between the three groups ( table 1 ) . serum ca concentration was significantly high in the cervical cancer group compared to that of healthy control group ( 9.51 0.01vs 9.44 0.01 , p = 0.000 ) . on the other hands , urine dpl concentration was significantly low in the endometrial cancer group in comparison with healthy control group ( 8.08 0.76 vs 8.45 0.05 , p = 0.000 ) ( table 2 ) . for each t - scores of l1 , l2 , l3 , l4 , femur neck ( fn ) , femur trochanter ( ft ) , the t - scores of basal bmd at l2 and l4 were significantly lower in patients with cervical cancer ( -0.62 0.07 vs -0.44 0.06 , p = 0.038 in l2 ; -0.65 0.08 vs -0.15 0.06 , p = 0.000 in l4 ) compared to those in the healthy control group . endometrial cancer group did not showed significant difference in t - scores compared with the healthy control group ( table 3 ) . additionally , the incidence of osteoporosis according to the basal status of bone mass was significantly higher in patients with cervical cancer ( 18.81% ) compared to that of healthy control ( 10.81% ) . the incidence of osteopenia was also significantly higher in cervical cancer group ( 38.99% ) compared with the control group ( 36.29% ) ( p = 0.16 ) . endometrial cancer showed the higher incidence of osteoporosis ( 16.47% ) and lower incidence of osteopenia ( 28.24% ) , but there was no statistical significance ( p = 0.228 ) . the dichotomization of the t - score according to the decreased ( t - score < -1.5 ) or relatively normal ( t - score -1.5 ) indicate more distinct results that cervical cancer group had higher risk of bone loss ( p = 0.020 ) in comparison with the endometrial cancer group ( p = 0.701 ) ( table 4 ) . no significant differences in clinical , laboratory , or bmd data were observed among patients with cervical cancer divided according to cancer stage ( table 5 ) . as the long term survival has become longer and prognosis has been improved in gynecologic cancers , the quality of life in cancer survivors is important , these days . to support the quality of life , bone health is essential , especially in older women . quality of life is increasingly important for long - term survivors of gynecologic cancers , and osteoporosis is one of the major quality - of - life issues among gynecologic cancer survivors . this study reveals that cervical cancer has higher risk of impaired bone health per se , and for the treatment of cervical cancer , osteoporotic aspect should be considered . cervical cancer ranks as the third most common cancer in women , and it is the second most frequent cause of cancer death among women 18 . there are numerous risk factors for cervical cancer including young age at first intercourse , multiple sexual partners , cigarette smoking , race , high parity , low socioeconomic status , and chronic immune suppression , whereas the association with oral contraceptive use is controversial . many of these risk factors are linked to sexual activity , and not to hormone status . in the present study , t - scores of basal bmd in l2 and l4 were significantly lower in patients with cervical cancer compared to those in controls , and the incidence of osteoporosis and osteopenia were significantly higher in patients with cervical cancer compared to that in controls . the association between cervical cancer and bmd of the lumbar spine has been addressed in a few studies . cho and colleagues 13 compared spinal bmd data measured by dual - photon absorptiometry ( dpa ) in 85patients with cervical cancer to the data in 148 control women and reported that the mean lumbar bmd in women with cervical cancer was 12.8% lower than that in the controls after adjusting for age and menopause duration . this was the first study that examined the association between cervical cancer and bmd ; however , it was limited by the fact that they used dpa , which was found to be less accurate than dxa for the measurement of bmd . hung and colleagues 14 reported that premenopausal patients with cervical cancer without bone metastases had significantly lower bmd ( 0.95 0.03 g / cm ) compared to that in controls(1.08 0.02 g / cm ) in the lumbar spine ( l2 - 4 ) . by contrast , lee and colleagues 19 reported that the spinal bmd inpatients was not statistically lower compared to that in controls , which is in disagreement with other studies including ours . several factors are related to the activation of osteoclasts by tumor cells including parathyroid hormone - like peptide 7 - 9 , transforming growth factor 10,osteoclast activating factor 11 , and prostaglandins 12,20 , and these osteolytic factors may contribute to the development of cancer - induced bone loss ( cibl ) . in patients with cancer , greater osteoclastic activity , markedly reduced osteoblastic surface , osteoid surface , and osteoid volume have been noted by quantitative histochemical studies of the bone 11,21 . in arat model , tumor - bearing rats showed a reduction in the volume of trabecular bone and an increase in the number of osteoclasts , which was presumably mediated by a humoral factor that activates existing osteoclasts and induces monocytes to differentiate into osteoclasts 22.biochemical markers of bone metabolism are indicators of both the formation and resorption of bone 23 . biochemical bone turnover markers provide clinically useful information about the normal and pathologic processes that reflect bone cell activity in the skeleton , and they can provide valuable insight into interactions between bone remodeling and tumors . in the present study , ca concentration in serum was higher in cervical cancer group . it can be explained by the possibility of higher bone resorption process in cervical cancer in contrast with endometrial cancer of healthy women . other significant biochemical marker , urine dpl , was lower in patients with endometrial cancer than in controls . dpl is one of two pyridinium cross - links that provide structural stiffness to type i collagen found in bones 24 , and it is used as a bone turnover marker along with other bone markers . it can be assumed that endometrial cancer , as the hormone - dependent tumor , may have high bone resorption like other several cancers , however , osteoblastic action may also be increased in the response with hormone . there are many bone turnover markers such as carboxy - terminal collagen crosslinks ( ctx ) , urine n- terminal collagen crosslinks ( ntx ) , amino pro - peptide of type 1 collagen ( p1np ) , and bone specific alkaline phosphatase ( bsap)6 . further study with these biomarkers in gynecologic cancer may give the clue for the diagnosis and management of cancer patients . if the reduced bone mass in the lumbar spine observed in the present study was related to the bone - resorbing factors , we would expect to see hypercalcemia in patients with cervical cancer , but all the patients in our study were normocalcemic . first , calcium reflux from bone may have been too subtle to be detected by the technique used . another explanation is that some cases of malignancy may have been associated with elevated levels of bone - resorbing material seven in the absence of hypercalcemia because of regulatory mechanisms that maintain normocalcemia , as proposed by henderson and colleagues 25 . in the femur neck and trochanter , we found no differences of bmd between patients with cervical cancer and controls , which are inconsistent with the results reported by lee and colleagues 19 , who showed that total femoral bmd in patients with cervical cancer was significantly lower compared to that in controls . endometrial carcinoma is the most common malignancy of the female genital tract in the usa . most of the risk factors for the development of endometrial cancer , such as nulliparity , late menopause , and unopposed estrogen therapy , are related to prolonged , unopposed estrogen stimulation of the endometrium , and several medical conditions leading to long - term estrogen exposure , such as polycystic ovary syndrome and functioning ovarian tumors , are associated with an increased risk for endometrial cancer 26 . to the best of our knowledge , lee and colleagues 15 retrospectively analyzed the bmd of the spine and femur using dxa in 31 patients with endometrial cancer without bone metastases and 61 controls who were treated with surgery for benign disease in korea . these authors reported no differences in the bmd of the spine or femur between patients with endometrial cancer and controls . in the present study , there were no significant differences of basal bmd in the lumbar spine and femur between patients with endometrial cancer and controls , and the levels of biochemical bone markers did not differ significantly between the two groups . osteoporosis is strongly related to the decline of endogenous estrogen level . on the other hand , high levels of endogenous estrogen are related to endometrial cancer , which can lead to increased bmd . previous studies have reported decreased risks of endometrial cancer among women with pre - existing bone pathologies such as low bmd , fracture , and osteoporosis 27-29.in a swedish cohort , a significantly reduced risk of hip fracture was observed in patients with endometrial cancer ( standardized incidence ratio ( sir ) 0.6 ; 95% confidence interval(ci ) 0.5 - 0.8 ) 27 . they reported that women diagnosed with osteoporosis in all age groups were at decreased risk of endometrial cancer ( sir 0.61 ; 95% ci 0.46 - 0.79 ) 29 . in the present study , neither bmd nor bone turnover markers were different between patients with endometrial cancer and controls . we hypothesized that bmd values at the lumbar spine and femur were not different between these groups because bone mass in patients with endometrial cancer may have reached a balance between the negative effect of cibl and the positive effect of high endogenous estrogen levels related to endometrial cancer . cancer - treatment - induced bone loss ( ctibl ) may cause bone fragility and an increased susceptibility to fractures , and bone loss occurs more rapidly and tends to be more severe in patients with ctibl than in those with normal age - related bone loss ; therefore , prevention , early diagnosis , and treatment of ctibl are essential to decrease the risk of fracture 30.ctibl is most common in patients with breast or prostate cancer who receive chemotherapy , hormone therapy , or surgical castration , as these can cause hypogonadism and induce bone loss . in women with gynecological malignancies , concurrent chemo - radiation therapy ( ccrt ) , particularly in patients with cervical cancer , has been observed to reduce bmd 31,32 . nishio and colleagues 31 reported that ccrt for cervical cancer significantly increases bone resorption marker levels and reduces bmd of the lumbar spine , particularly in patients with cervical cancer who have received ccrt . hwang and colleagues 32 reported that the lowest t -scores for bmd were significantly lower in the women with cervical cancer treated with ccrt compared with the control women , and the serum total alp level was also significantly higher in women with cervical cancer treated with ccrt . in the present study , the t - scores of the basal bmds of all lumbar vertebrae were not significantly different between patients with cervical cancer and controls in l2 and l4 , not in femur . hwang and colleagues 32 reported that the bmds of all vertebrae except l4 were not significantly different between patients with cervical cancer treated with ccrt and controls . although this study shows the bmd status before the treatment such as surgery and ccrt , lumbar spine in patients diagnosed with cervical cancer should be evaluated because the risk of bone loss is high due to cancer itself and further treatment . in those patients , anti - osteoporotic treatment such as ca supplementation or bisphosphonate although our study had a larger sample size compared to those in other studies that examined bmd in patients with gynecologic cancer , the limitations of this study mainly stem from its retrospective study design . the inclusion of confounding risk factors for bmd such as smoking , alcohol , dietary differences , vitamin d levels , and physical activity may have provided a clearer association between gynecologic cancer and osteoporosis . especially , serum 25 hydroxy - cholecalciferol as vitamin d may be altered in patients in cancer . further study considering those limitation scan provide us the more distinct relationship between cancer and bone health . in summary , women with invasive cervical cancer have a lower bone mass density and are at increased risk of osteoporosis in the lumbar spine before anticancer treatment . there were no differences in bmd at any site between patients with endometrial cancer and controls . further prospective large scale trials are needed to clarify the association between the gynecologic cancers and bone mass density . respiratory distress continues to account for significant mortality and morbidity in the neonatal intensive care unit . at birth , respiratory distress syndrome ( rds ) in premature infants is caused by a structural immaturity of lungs and the insufficient production of surfactant and its incidence is inversely related to gestational age . the problems concerning to the respiratory system prolonge the hospitalization period in the premature infants . endothelin ( et ) is a peptide of 21 amino acids in chain with two disulfide bonds with three distinct isoforms : et-1 , et-2 , and et-3 . endothelin causes isolated contraction of pulmonary veins , vascular smooth muscle mitogenesis , myocardial cell hypertrophy , positive innotropic and chronotropic effects , bronchoconstriction , mucous secretion , cellular proliferation , and inflammatory reactions . hypoxia , stress , antidiuretic hormone , and the secretion of some mediators stimulate its synthesis . clinical investigations have shown increased plasma concentrations of et-1 during rds and in case of pulmonary hypertension of other origins . but it is unclear whether et-1 is actually responsible for the pulmonary hypertension , or the increased et-1 plasma concentration is a result of the pulmonary hypertension that originated otherwise [ 58 ] . also transforming growth factor beta ( tgf- ) is a family of three isoforms that regulate cell growth and differentiation , extracellular matrix sythesis cytokines production , and vascular neogenesis . the increase in tgf- precedes the development of pulmonary hypertension which increases circulating et-1 levels in animals . tgf- induces et-1 gene expression and et-1 peptide synthesis in bovine pulmonary artery endothelial cells [ 10 , 11 ] . the cells responsible for increased et-1 sythesis during hypoxia are unclear , and short - term effects of hypoxia raise plasma et-1 levels in animal models ; whether chronic hypoxia would lead to different results is unknown . experimental studies have suggested that et-1 plays an important role in pulmonary vascular reactivity in neonatal rds . there is also an elevation of et-1 in tracheal aspirates from these infants . tgf- showed the strongest stimulatory effect on et-1 and gene transcription in vascular smooth muscular cells . there are few studies measuring et-1 and no study measuring tgf- by enzyme immunoassay with a very small number of human premature newborns suggesting that et-1 is elevated in rds . the purpose of this present study is to evaluate the serum levels of et-1 and tgf- in the newborns with respiratory distress ( diagnosis as rds , as transient tachypnea ( rds-2 ) , and as meconium aspiration syndrome ( mas ) ) , to investigate the meaningfulness of the repetitive values of et-1 in the followup of these diseases , and to determine the reflection of serum et-1 level on the mortality at the first six hours after birth . in this study , newborns with respiratory distress hospitalized into the newborn intensive care unit were included . the study group was evaluated by 100 newborns , 62 diagnosed as rds , 24 as rds-2 , and 14 as mas within the last six months . moreover , a control group was evaluated with 20 healthy newborns . for the study , written permits were taken from the parents of each newborn , as well as an approval of the regional ethics comittee . the gestational age of the newborns was determined according to the new ballard score . according to the gestational age , newborns younger than 38 weeks were classified as premature and newborns between 3842 weeks as mature . after a detailed physical examination , the newborns were investigated with respect to their blood gases , complete blood count , full blood biochemistry , and c - reactive protein levels , and their culture samples were taken . moreover , first blood samples were obtained from all premature and mature sick or healthy newborns in the first six hours after birth . the serum was collected into polypropylene tube and centrifuged immediately and was stored at 70c . in addition , repetitive blood samples were obtained at the third , 7th , 14th , and 28th day from the patients whom oxygen supply continued . these serums were also stored at 70c . then , the samples were resolved and they were studied with elisa method in the immunology laboratory . plasma tgf- level was determined by capture elisa according to the instructions of r & d systems using monoclonal antihuman tgf- , r & d systems , inc ( usa ) . in brief , 100 l of the capture antibody was transferred to an elisa plate and incubated overnight at room temperature . after removal of the buffer , the plates were blocked by adding 300 l of pbs containing 5% tween 20 , 5% sucrose , and 0.05% nan3 to each well and incubated at room temperature for a minimum of 1 hour . 100 l of blood plasma sample per well was added , the elisa plate was covered with an adhesive strip and incubated 2 hours at room temperature . 100 l of streptavidin hrp ( r & d systems , catalog # dy998 , 1/200 in appropriate diluent ) was added to each well ; the plate was covered and incubated for 20 minutes at room temperature . after subsequent addition of substrate solution and stop solution ( both from r & d systems , inc ) , the optical density of each well was determined within 30 minutes , using a microplate reader set to 450 nm . et-1 was determined by an enzyme immunoassay ( quantiglo human et-1 , r & d systems , inc , minneapolis , minn , usa ) . the minimum detectable dose of et-1 was 0.16 pg / ml , with intra- and interassay coefficients of variation of 2.5 and 5% , respectively . moreover , in the differential diagnosis the meaningfulness of the et-1 and tgf- values was investigated using the scheffe and tukey post hoc tests , in the followup , the differences of the et-1 levels were investigated using the kruskal - wallis variance analysis . in all of the results the newborns comprimising the study group had a gestational age between 2842 weeks . from all of the patients 62 ( 62% ) newborns of 24 newborns with rds-2 diagnosis 16 ( 67% ) were mature and of 14 newborns with mas diagnosis 11 ( 79% ) were mature . in the control group , only 11 ( 55% ) newborns were mature ( table 1 ) . male predominance with a percentage of 61% was determined in the group with rds , and weight results were considerably lower due to prematurity ( 1367 368 g ) . plasma endothelin-1 and tgf- concentrations of the mature newborns in the control group were measured as 0.77 0.56 pg / ml and 0.25 0.41 pg / ml , respectively , and did not show a significant difference according to the gestational age ( p > .05 ) ( figure 1 ) . plasma endothelin-1 and tgf- concentrations of the newborns with different diagnosis measured in the first six hours of life are summarized in figure 2 . in the description of the effectiveness of the treatment and in the early determination of the prognosis plasma et-1 and tgf- concentrations in newborns with mas diagnosis were determined as 5.70 5.87 pg / ml and 3.75 1.94 pg / ml , and with rds diagnosis were determined as 3.37 1.59 pg / ml and 2.05 0.98 pg / ml . according to the control group , plasma et-1 and tgf- concentrations of the newborns with respiratory distress were determined to be statistically higher ( p < .05 ) . with the oxygen supply , it was observed that plasma et-1 concentrations of the newborns obtained in the first six hours of life are desposed to decrease regularly in the following days ( figure 3 ) . in the same way , plasma et-1 concentrations obtained in the first six hours and repetitive plasma et-1 concentrations in the following days were statistically higher in the newborns who died later compared to the survivors ( p < .05 ) ( figure 4 ) . respiratory distress is a major problem in the newborns and different reasons cause this problem . among those , the most frequent ones are rds ; a problem of premature newborns , and the others are rds-2 and mas ; a problem of mature newborns . in this study , diseases causing respiratory distress after birth in the early period were evaluated , and rds was the most frequent ( 62% ) . endothelin-1 and tgf- levels , the vascular factor , and material of respiratory distress were investigated . in the control and patient groups ' plasma , et-1 and tgf- concentrations were statistically different . the highest value was obtained in the newborns with mas and the others were ordered as rds , rds-2 , and the healthy newborns . kaapa et al in a similar study found that plasma et-1 concentrations were not correlated with the pulmonary pressure but that high plasma concentrations of et-1 reflected severe pulmonary damage . in another study , there was a significantly higher et-1 concentration in newborns with pulmonary hypertension than healthy newborns or newborns with rds . in contrast of our study , kuo et al determined the highest values of plasma et-1 concentrations in the first six hours of life in the newborns diagnosed as rds , and the newborns diagnosed as mas had a second highest values of et-1 concentrations . like our study , kojima et al found out that plasma et-1 concentrations in newborns with rds were higher compared to the newborns with rds-2 . a study of benjamin et al demonstrated that infants with and without rds had similar umbilical cord et-1 concentrations , whereas et-1 concentrations were higher in rds than in control newborns 1840 hours after birth . the increased vascular resistance in rds may be related to high plasma et-1 concentrations . in an experimental model of rds in the newborn lamb , the et-1 concentration was increased after induction of rds concomitant with the development of pulmonary hypertension , from an early time point onwards . increased et-1 concentration during rds appeared to be reached in the early phase of pulmonary hypertension development . also increased circulating levels of et-1 were correlated with the severity of pulmonary hypertension . whereas , tgf- is secreted from the alveolar macrophages in the lungs , and in case of damage it is responsible to the organization of the fibrosis growth , inflammatory response , and the recovery of the tissue . for this reason , the tgf- studies were realized in the patients with bronchopulmonary displasia , where fibrosis was dominated [ 23 , 24 ] . in our study , according to the results of plasma et-1 levels , the first highest levels of tgf- were in newborns with mas and the second were in newborns with rds . starting from the moment of the diagnosis , it was observed that et-1 concentrations of the patients who received surfactant and mechanic ventilator supply were decreased . kuo et al and niu et al emphasized those plasma et-1 concentrations of the newborns with and without bronchopulmonar dysplasia did not show any difference . the endothelium modulates vascular tone by releasing endothelium - derivated vasodilatators , including nitric oxide , prostacyclin , bradykinin , and vasoconstrictors such as et-1 and angiotensin ii , in response to a number of biochemical and physical stimuli . recent studies have suggested that an imbalance between nitric oxide and et-1 may contribute to changes in vascular tone observed in these diseases . a number of vasculopathies associated with an impaired bioavailability of nitric oxide have been found to be linked to enhanced sythesis of et-1 . in our study , plasma et-1 concentrations might have a best indicator of the prognosis in the first six hours of life , but tgf- concentrations did not have the same effect . because it was a significant difference between the survivors and dead newborns , in whom et-1 concentrations were higher in the first six hours of life . these newborns presented severe damage in the lungs , starting from the first hour . we did not see any other study emphasizing this subject in the literature . as a result , it was decided that , in the differentiating diagnosis of the rds , rds-2 , and mas , which are a significant problem of premature and mature newborns , the investigation of et-1 and tgf- concentrations is meaningful , but that in wider groups , it is required to determine the borderline values . it was observed that the et-1 levels investigated in the first six hours are more useful in determining the prognosis , and the et-1 concentrations investigated in the following days are more meaningful presenting clinical recovery . in the determination of prognosis , tgf- concentration invesitigated in the first six hours does not seem meaningful . since the results are still contradictory , it was emphasized that it is required to carry out new researches . plasma et-1 concentrations of the healthy premature and mature newborns were 0.79 0.44 and 0.77 0.56 pg / ml , respectively . also tgf- concentrations in this group were 0.17 0.37 and 0.25 0.41 pg / ml , respectively , and was not significantly different ( p > .05 ) . et-1 concentrations according to diagnosis in the sick newborns at the sixth hour after birth were 3.37 1.59 pg / ml in rds , 1.60 0.66 pg / ml in rds-2 , 5.70 5.87 pg / ml in mas , and 0.78 0.50 pg / ml in healthy group . only the concentrations of et-1 in rds and mas groups were significantly different ( p < .05 ) . also plasma tgf- concentrations were 2.05 0.98 pg / ml in rds , 1.59 0.66 pg / ml in rds-2 , 3.75 1.94 pg / ml in mas and 0.22 0.39 pg / ml in healthy group . only the difference in rds and mas group was significant ( p < .05 ) . changes of the plasma et-1 ( pg / ml ) concentrations during the oxygen treatment days in the different dignostic newborns tended to decrease . the plasma et-1 concentrations in the sixth hour of life were decreased at third day as 2.801.49 pg / ml in rds , 1.28 0.84 pg / ml in rds-2 , and 3.69 2.13 pg / ml in mas group . et-1 concentrations in the newborns who died were 4.44 1.26 pg / ml in rds and 11.60 8.97 pg / ml in mas groups ( p < .05 ) and in those who survived were 2.42 1.19 pg / ml in rds and 3.41 1.31 pg / ml ( p < et-1 concentrations in the newborns who died were 3.70 1.31 pg / ml in rds and 7.30 0.00 pg / ml in mas group ( p < .05 ) and in those who survived were 2.04 1.20 pg / ml in rds and 2.77 0.73 pg / ml ( p < .05 ) at the third day of life . et-1 concentrations in the newborns who died were 3.42 1.27 pg / ml in rds and 7.00 0.00 pg / ml in mas group ( p < .05 ) and in those who survived were 1.82 1.15 pg / ml in rds and 1.60 0.00 pg / ml ( p < .05 ) at the seventh day of life . et-1 concentrations in the newborns who died were 3.03 0.95 pg / ml and in those who survived were 1.60 1.35 pg / ml in rds ( p < .05 ) at the 14th day of life .

objective : to evaluate the bone mineral density ( bmd ) in the lumbar spine and femur in postmenopausal women with cervical cancer and endometrial cancer without bone metastasis in comparison with that in healthy control postmenopausal women , and to assess the loss of bmd according to the cancer stage.materials and methods : we analyzed the bmd of the lumbar spine and femur using dual - energy x - ray absorptiometry ( dxa ) in 218 patients with cervical cancer , 85 patients with endometrial cancer , and 259 healthy controls . the serum levels of calcium ( ca ) , phosphorus ( p ) , osteocalcin ( osc ) , and total alkaline phosphatase ( alp ) , and urine deoxypyridinoline(dpl ) were measured in all participants.results : age , body mass index , parity , and time since menopause were not significantly different between the three groups . serum ca level was higher in the cervical cancer group ( p = 0.000 ) , however , urine dpl was lower in endometrial cancer group ( p = 0.000 ) . the t - scores of basal bmd at the second and fourth lumbar vertebra ( l2 , l4 ) were significantly lower in patients with cervical cancer ( p = 0.038 , 0.000 , respectively ) compared to those in the healthy control groups . additionally , the incidence of osteoporosis and osteopenia basal status of bone mass was significantly higher in patients with cervical cancer compared to that in controls ( p = 0.016 ) . no differences in basal bmd of the lumbar spine and femur were observed between patients with cervical cancer according to their stages.conclusion : our results suggest that postmenopausal women with cervical cancer have a lower bmd and are at increased risk of osteoporosis in the lumbar spine before receiving anticancer treatment compared with postmenopausal women with endometrial cancer . the purpose of this present study was to evaluate the serum levels of et-1 and tgf- in the newborns with respiratory distress . in this study , newborns with respiratory distress hospitalized into the newborn intensive care unit were included . the highest values of et-1 and tgf- were obtained from newborns with diagnosis as meconium aspiration syndrome ( 5.70 5.87 pg / ml and 3.75 1.94 pg / ml , resp ) in the sample obtained in the first six hours after birth , and these are statistically different from control group ( p < .05 ) . also , same results were obtained for newborns with respiratory distress syndrome ( 3.37 1.59 pg / ml and 2.05 0.98 pg / ml , resp ) . after oxygen treatment , et-1 values obtained in the first six hours of life were decreased regularly in the following days ( p < .05 ) . in the differentiating diagnosis of the respiratory distress of newborns , the investigation of et-1 and tgf- levels is meaningful . the et-1 levels investigated in the first six hours is more useful in determining the prognosis , and repeating et-1 levels in the following days is more meaningful to determine clinical response .

thyroid cancer is the 10th commonest cause of cancer in russian women , and the age - specific incidence of thyroid cancer has been on the rise . while most thyroid cancers have a good prognosis , those that show tracheal invasion often show poor survival because of extensive resection or tumor recurrence . surgical intervention is the key point of treatment , and appropriate surgical planning is essential to get a promising prognosis , postsurgical quality of life , and social rehabilitation of a patient . despite an increasing role of nonadjuvant radiotherapy and/or chemotherapy in the treatment of thyroid cancer , the surgical approach is still used when recurrence is treated after radiotherapy / chemoradiotherapy or in the case of combined treatment of patients with thyroid papillary carcinoma invading the trachea . the extensive resection that may be necessary for locally invasive thyroid cancer has led some surgeons to attempt to conservatively approach these tumors using peeling or shaving techniques aiming at preserving function [ 2 , 3 ] . such conservative approaches rely on the administration of postoperative radioactive iodine with / without external beam radiotherapy to manage a microscopic disease . many patients with locally advanced thyroid cancer tend to be older patients with more poorly differentiated histologic variants . tumors in such patients can be nonradioactive iodine avid , and the response to radioactive iodine therapy can be disappointing . recently , the importance of sparing surgery has also increased in clinical head and neck oncology . it fully applies , for instance , to the treatment of patients with laryngeal cancer . better functional and cosmetic result for a patient should also be an objective . in this report , we present a patient with advanced thyroid papillary carcinoma involving the trachea , who has previously undergone total thyroidectomy followed by radioactive iodine therapy in 2012 . the first one was a window resection of the trachea followed by an open tracheostomy , and the formed knitted tini - based mesh endograft ( ktnme ) , as reported by muhamedov et al . the second stage was the reconstruction of the large tracheal defect with the harvested endograft and the skin draping over the wound . the patient presented here is a 54-year - old female who was referred and admitted to the hospital of the tomsk cancer research institute on april 2 , 2014 ( clinical case # b273xx ) . the patient underwent total thyroidectomy for papillary thyroid cancer ( type t4nxm0 ) in june 2012 when the cancer directly involved the tracheal wall , followed by radioactive iodine therapy in the vladivostok central clinical hospital in july 2012 . apparently , she was considered with no evidence of disease until december 2013 when the mass in her trachea that she noticed had been gradually increasing in size within the recent months , and the histological findings verified well - differentiated papillary carcinoma recurrence . there were objectively no remarkable signs except for a neck incisional scar and the subcutaneous , rounded , newly grown palpatory mass detected ( about 1.5 cm in diameter ) . she had other symptoms such as dyspnea at rest , hoarseness , and minor hemoptysis . fiberoptic endoscopy and magnetic resonance imaging ( mri ) revealed a circumferential stenosis on the right side below the vocal cord , invading the lumen of the trachea . a submucosal mass from the 2nd to the 5th tracheal ring with an irregular surface penetrating the mucosa with signs of ulceration and erosion was noted ( fig 1 ) . according to the above findings , the case was classified as t4n0m0 , and a double - stage surgical treatment was scheduled ( fig 2 ) . the first stage was performed on april 15 , 2014 . to involve the platysma muscle , the u - shaped incision was made alongside the postoperative scar ( fig 3a ) . subcutaneous fat was removed with electrocautery to aid in exposure and to prevent later fat necrosis . dissection proceeded through the platysma until the midline raphe between the strap muscles was identified ( fig 3b ) . the sternocleidomastoid muscle was thus separated , retracted laterally , and preserved . by tracing , the right recurrent laryngeal nerve was assessed to be intact and half of the trachea ( 5.5 2.3 cm ) was dissected including 5 cartilaginous rings together with the left recurrent laryngeal nerve . the tumor was removed together with the invaded paratracheal tissue bilaterally ( fig 3c ) . the medial edges of the sternocleidomastoid muscle were sutured to a rectangular tracheal opening . to avoid tracheal stenosis , the anterior side of the defect was open , and a stent tube was kept in place . for muscle - flap prefabrication , the prepared ktnme , as described by muhamedov et al . the cut edges of the tracheal opening were circumferentially overedged to the skin with a few absorbable sutures to facilitate cannulation . after the airway was confirmed intact based on co2 return and bilateral breath sounds , the tracheostomy tube was secured to the skin with 40 sutures ( fig 3e ) . to avoid the risk of subcutaneous emphysema and subsequent pneumomediastinum , the skin was not tightly closed . a sponge soaked with iodine between the skin and the flange was placed for 24 h to deflect infection and anxiety about minor oozing of the skin edge . the patient was extubated after 7 days , and there was no granulation tissue or stenosis of the tracheal lumen 3 weeks thereafter ( fig 4 ) . the reconstructive stage was performed on may 19 , 2014 . the o - shaped incision around the formed tracheotomy having the gap 1.5 cm from the right edge was made . a cellulocutaneous flap was separated and overturned so that the epidermis overlapped the tracheostomy lumen ( fig 5a ) . the harvested ktnme after prefabrication was separated with the pedicle flap of the medial edge of the sternocleidomastoid muscle ( fig 5b ) , placed over the trachea , and sutured to the trachea ( fig 5c ) . the remaining defect in the anterior part of trachea was closed without any special procedure . the sliding skin flap was swung over the wound followed by aesthetic wound closure to achieve optimal cosmetic results with minimal scarring ( fig 5d ) . postoperative bronchoscopy revealed a pink - coated part in the tracheal mucosa with no stenosis ( fig 5e ) . the patient was discharged on june 3 , 2014 , and referred back home to be supervised by a local hospital , where she was socially rehabilitated in full . the patient remains alive and without any evidence of recurrence or complaints well over 24 months after surgery . she continued her job 2 months after discharge and calls us every 6 months to confirm her health status and current matters . the possibility to perform a sparing surgery depends on several factors , including the availability of adequate material for the tracheal replacement after lesion [ 7 , 8 ] . however , functional outcomes of such a kind of the treatment do not always satisfy both patients and clinicians given that a large portion of the trachea is to be resected in locally advanced thyroid papillary carcinoma invading the trachea . some surgical methods , on account of the improvement , standardization , and more widespread use of reconstructive surgery with microvascular flaps , for the management of tracheal invasion by thyroid carcinoma have been reported in the literature , so far with no consensus on the best approach . the development of techniques and the selection of an optimal material for the tracheal reconstruction remains the urgent aim of modern head and neck surgical oncology . the brief overview of published articles on tracheal sparing surgery studies reflects , in fact , that there are no commercially produced implants for a wide clinical practice in cases of extensive lesions of the trachea . since it is a mobile organ when breathing , swallowing , speaking , eating etc . , it is critical to raise standards regarding the material selection for the replacement . to succeed , clinicians should make their unconventional choice of what approach to use and how to perform the correct reconstruction . as a rule , the structures involved in cases of invasive thyroid papillary carcinoma are mostly the trachea , larynx , laryngeal nerve , and other surrounding sites . since a regional spread , increased incidence of local recurrence and distant metastasis are common ways of invasion to the surrounding structures , it is crucial to control local invasion , which makes treatment relatively difficult . the management of locally invasive , well - differentiated papillary carcinoma of the thyroid is disputable . palliative surgery had been the most common surgical procedure used for the treatment of patients with thyroid carcinoma invading the trachea for many years . however , the shaving - off procedure was reported to be often inadequate , and the postoperative survival rate was much worse in patients undergoing partial resection than in those undergoing total resection . for adequate surgery , as in our case , accurate preoperative examination of the tracheal invasion including bronchoscopy , ct , and mri is required to clarify the involvement of adjacent organs . the generally accepted technique for the reconstruction of circular tracheal lesions is to form the end - to - end primary anastomosis between the proximal and distal margins of the defect . however , this is feasible for small ( less than 3 rings ) circular defects , and a larger area of invasion may limit the feasibility of partial tracheal resection because it may lead to kinking or stenosis of the trachea after repair . contraindications include extensive involvement of the trachea , such that 6 or more tracheal rings need to be resected or simultaneous invasion of the esophagus occurs . moreover , in cancer patients , the upward trend in postoperative complications is probable , including a failure of the anastomosis or scar stenosis due to a previous treatment and the weak reparative ability of the tissue . in case of extensive tracheal defects ( more than 5 cm ) , the most reliable method is reconstructive surgery , which involves a layerwise restoration of the tracheal wall . such an approach assumes a fusion of the framework and a thin flap , which adequately replaces the frame function of the trachea without narrowing the lumen . there are several similar studies that aimed at exploring tracheal reconstruction considering the frame function , but they are strictly experimental and still not applicable in cases of large defects of the trachea . it should be pointed out that applying the implant in large defects of the trachea has to be done without any contact with the lumen when there are signs of a primary inflammation . it is suggested to ensure strict adherence to the criteria , otherwise the inflammatory process that occurs in situ eventually affects the consistency of the graft and leads to granulation tissue growth and a failure of the whole reconstruction stage . in summary , in order to succeed in a complex sparing surgery , the obvious procedure is to use the prefabricated endograft covered with a thin - walled lining by the adjacent tissues , as we did . this technique implies 2 stages of reconstruction , which do not contravene the modern concept of sparing surgery as repeated corrective treatments to restore the lumen are necessary . a two - stage surgical treatment has been applied before in cases of large tracheal lesion defects : first , after resection of the tumor , the tracheal wall and skin are sutured for the development of a persistent tracheocutaneous fistula trough. second , delayed closure of the tracheal defect was performed . unfortunately , compared to our method , this technique involves long - term treatment and is also not always aesthetically satisfactory because of the need for harvesting a local flap . in the case described herein , we demonstrated that the prefabricated ktnme is a useful and simple technique of sparing surgery for the tracheal reconstruction after excessive window resection in a patient with advanced thyroid papillary carcinoma invading the trachea . the offered method of a two - stage sparing treatment leads a long palliative period associated with a good quality of life . besides the ktnme applied can adequately restore the frame function of the trachea without postoperative aggravation . as a consequence of the successful application , the ktnme is shown to provide a superior reconstruction and stability for the tracheal grafting during the implantation procedure and thereafter . the surgery was performed in accordance with the ethical principles outlined in the declaration of helsinki . its protocol was approved by the ethical committee of the tomsk cancer research institute , and informed consent was obtained from the patient before the upcoming intervention . the authors declare that there are no conflicts of interests regarding the publication of this paper . the history of cross - cultural exchanges between the people of india and their colonial rulers provides a fascinating insight into how these encounters shaped medicine and medical education in india . given the backdrop of colonialism and hegemony , the confluence of the east and the west was turbulent . in the 16 century , it was the portuguese who first introduced western medicine into india . in 1600 , the medical officers who arrived along with the east india company 's first fleet of ships also brought western medicine in india . initially , medical departments , with surgeons , were setup to provide medical relief to the troops and employees of the east india company . in 1775 , these were essentially constituted by staff of the commander - in - chief of the british indian army in each presidency . medical departments were setup in bengal , madras , and bombay presidencies in 1785 , and these looked after both military personnel and british civilians . the mutiny of 1857 led to the dissolution of the east india company and the british government was established in india . several organized services such as the indian medical service , the central and provincial medical services , and the subordinate medical services were initiated to provide medical services and improve public health . a public health commissioner and a statistical officer were also appointed to the government of india . in 1869 , the medical departments in the three presidencies were amalgamated into the indian medical service . a competitive examination was conducted in london to recruit people into the indian medical service . the european officers of the indian medical service headed the military and civil medical operations in the three presidencies . however , they needed trained assistants and supporting staff such as apothecaries , compounders , and dressers in their work . appointing european doctors had large financial implications . this prompted the british government to look toward establishing a system of medical education in india to recruit local staff . in 1822 , european texts in anatomy , medicine , and surgery were translated into the local languages for the benefit of students . though dissection was not performed , clinical experience in different hospitals and dispensaries was mandatory . john tyler , an orientalist , was the first superintendent of the native medical institution . classes on unani medicine were held at the calcutta madrasa , while the sanskrit college conducted classes in ayurvedic medicine . successful native graduates were absorbed into government jobs . in 1826 , to offer indians the opportunity to learn and practice western medicine , an indian medical school was started in southern bombay with surgeon john mclennan as the superintendent . , the anglicists managed to overturn several cultural educational policies started by the vernacularists and orientalists . charles trevelyan , an ardent westernizer , chastised the british policy of educating europeans in the languages and cultures of the east and recommended that the asiatics ought to be educated in the sciences of the west . in 1833 , lord william bentinck appointed a committee to look into the state of medical education in bengal and the teaching of indigenous systems of medicine . in 1834 , the report , submitted by the committee led by dr . john grant , criticized the medical training and assessment conducted by the native medical institution . the report recommended that the state should found a medical college for the education of natives . the various branches of medical science should be taught to students , as in europe . the trainees should be able to read and write in english , bengali , and hindustani , and must be proficient in arithmetic . in february 1835 , thomas macaulay composed a powerful minute recommending that the government withhold further grants to institutions , conferring instruction in the native languages . the termination of official patronage to indigenous systems of medicine sealed the fate of the students of the two leading oriental institutions in calcutta . the classes held at the madrasa and the sanskrit college were discontinued in 1835 . in their place , a new medical college was established to train indian students in strict accordance with the mode adopted in europe through the medium of the english language . calcutta medical college was established in 1835 and it ushered in a new beginning to medical education in india . youths between 14 and 20 years of age were trained in the principles and practices of medical science using methods of the west . they were to be trained for a period not less than 4 years and not more than 6 years , after which they had to appear in a final examination . native doctors , and allowed to enter public service with an initial pay of rs . 30 a month . in bombay , sir robert grant became the governor and was deeply moved by the vast number of indians who died due to lack of proper medical care . he envisaged the idea of training indians in western medicine and as he tried to expedite his agenda for a more systematically planned medical college , he met with strong opposition . to quell this opposition and also to encourage a spirit of scientific inquiry , the medical and physical society of bombay was formed in 1835 . charles morehead studied the reasons which led to the abolishment of the previous medical school . in 1837 , the society concluded that establishment of a medical school was necessary for the education of the indians of the presidency in medical science to the extent of qualifying indians to become useful and safe practitioners of medicine . dr . charles morehead wrote that in gifting medical science to the people of india , there was a scope not merely for the operations of successful imitation but also for the adaptations of original genius . the college was not designed in imitation of the medical college in calcutta which intended to produce government servants . the college in bombay was designed to produce independent medical practitioners from the natives of india . in march 1838 , a generous grant by philanthropist sir jamsetjee jeejebhoy made way for building a new general hospital . the east india company endorsed the proposal to setup a medical college on july 18 , 1838 . the foundation stone of the grant medical college was laid in bombay in march 1843 with an aim to impart the benefits of medical instruction to the natives of western india through a systematic system . the general hospital which was opened in 1845 is now known as the sir jj hospital . the first - batch students who entered grant medical college were between the ages of 16 and 20 years . indians could undertake a course to qualify as doctors as assistant surgeons or they could undertake shorter courses to allow them to practice as medical subordinates ( sub - assistant surgeons , hospital assistants , and sanitary inspectors ) for british government services . afford better means of instruction in medicine and surgery to the indo - british and native youths , entering the medical branch of the service in the presidency . different courses were conducted for the medical apprentices of the apothecary branch and for pupils of the native branch of the military sub - medical department to be appointed as dressers later . later , midwifery , physiology , ophthalmology , and chemistry were added , and the course was extended to 3 years . eventually , medical colleges were started in other provinces too , with the purpose of producing a cadre of doctors who could be recruited into the subordinate medical services . in 1840 , the portuguese started the medicine and pharmacy licenciates , now known as goa medical college . university - affiliated medical education became the norm in the 1850s , after the opening of the first three indian universities in madras , bombay , and calcutta . madras medical college was the first in india to open its doors to women students in 1875 . even so , in 1877 , among the 8000 medical practitioners , only 450 were trained in western medicine . while nationalism provided a fertile soil for the revival of ayurveda and other indigenous branches of medicine , the demand for swaraj or home rule entailed that india needed to be projected as a progressive , modern , and scientific nation . therefore , the revivalist efforts during this period placed importance on establishing the scientific and progressive credentials of ayurveda . a proliferation of books on ayurveda in english , sanskrit , and vernacular languages tried to transform the hitherto relatively inaccessible knowledge into social knowledge as well as a shared system of knowledge among the practitioners . ayurvedic practitioners organized themselves and founded the all india ayurvedic congress . the themes central to the discourses at these conferences were british orientalism , the synthesis of medical systems , and the institutionalization of ayurveda . m. m. gananath sen , an ayurvedic practitioner from bengal , founded a college for the study of ayurveda and a pharmaceutical concern for manufacturing ayurvedic medicine . the bright spots for medicine during colonial times were the initiation of public health measures , vaccination , and the elevation of tropical diseases to a special area of study . a number of epidemiological and research studies were conducted on cholera , plague , malaria , tuberculosis , and leprosy . british india was a cauldron of several systems of medicine which intermingled with each other . long before the east india company entered the subcontinent , an exchange of knowledge and ideas had occurred between the indian systems of medicine and their foreign counterparts such as the arabs and chinese . the vaidyas and the hakims from the ayurvedic and unani systems of medicine complemented and borrowed from each other . the practitioners of these systems collaborated and learned from each other , and there is hardly any account of animosity between them . however , the interaction between the practitioners of indigenous and western medicine was anything but smooth . there was a clash of cultures where the east was seen as weak against the powerful knowledge of the west . both groups tried to differentiate their own set of ideas from those of the other . in the east , medicine was largely pluralistic and there was awareness and acceptance of alternative traditions . medicine was not viewed simply as a biological phenomenon and emphasis was given to a patient 's societal standing , environment , and relation with the therapist . as colonial arteries hardened , claims of the western superiority and scientific authority isolated western medicine . allopathic practitioners saw themselves as modernizers and often treated their indigenous counterparts with contempt for their inferior knowledge . while western medicine was accorded the status of official medicine , the state turned discriminatory and hostile toward the other systems . the rising tides of nationalism also posed to be an obstacle in a healthy exchange of ideas . the intrusion of western medicine was resented by the practitioners of indigenous medicine , and they stoutly defended their traditions . these practitioners also tried to avoid humiliation by acquainting themselves with the new techniques of diagnosis . in the 1920s , benaras hindu university developed a course which had both ayurveda and western medicine . the principal 's argument was when these are once installed , the students will see how ridiculous ayurvedic medicine is and it will die a natural death . if it were opposed , it would occupy a martyr 's place and be much more likely to continue . as panikkar points out , they were inclined to borrow , but they could not create a dialogue between the two epistemics . graduates of the government indian medical school were considered enabled to bring to bear on the problems of health and ill - health not only the expert knowledge of their own systems but , as far as practicable , the best that is in other systems also . these graduates sought to distance themselves from the graduates of madras ayurvedic college because the latter were seen as purists and therefore not attuned to modern methods . b class practitioners , against the a class accorded to medical graduates . the denial of registration to practitioners of indigenous systems of medicine by the madras medical registration act of 1914 was seen as gross discrimination . according to roger jeffery , indigenous practitioners who served about eighty percent of the people of the land were being treated as untouchables of the profession by the allopathic practitioners who considered themselves as age - old practices were followed blindly by their proponents with absolutely no scope for questioning and scientific scrutiny . this absence of openness to criticism and scientific rigor still is a cause for concern about scientific communities . however , pushed to a defensive position , knowing that western medicine was here to stay , there were several voices of reason , asking for cooperation and synthesis between the two systems of medicine . c. g. mahadeva argued , there can not be water - tight compartments between the two systems of medicine . an anonymous article in the june 1928 issue of the journal of ayurveda or the hindu system of medicine argued , medical education in india should be so devised that it should take into account not only the present - day medical education but also medical knowledge of the past while ayurveda can not move on in [ an ] old groove , allopathy should not be accepted in toto for india . while we should absorb the pathology of the seed of disease from allopathy , we must give the pathology of the soil in disease to modern medicine . the two angles are at present different but should be harmonized . in this period , when indigenous medical traditions were marginalized , the local doctors tried to secure a space within the newly created professional structure to attain a privileged social status . practitioners of indian systems of medicine are treated as second - class doctors in india even today . along the way , we have often ignored non - western concepts of disease , and discarded alternative ways of providing succor to humanity . however , even today , traditional medicine seems to dominate the primary levels of health care while western medicine is more popular as we move up the social ladder . having said this , much needs to be done to improve the quality and duration of training given to students of indigenous systems of medicine , including emphasizing the need for periodic curriculum review and improvement , adoption of scientific rigor and the spirit of inquiry . medical education in post - independent india faces significant challenges . these include the rapid , asymmetric rise in the number of medical schools , the questionable validity of student selection policies , a curriculum that is far removed from national health care requirements , and declining quality of teaching in medical schools . six decades after independence , educationists have still been unable to convincingly shrug off the colonial yoke . strangely , the curriculum followed by medical trainees has not been fundamentally altered since the days of the raj . the shrivastav committee advocated reorientation of medical education by national priorities and needs . in 1986 , the bajaj committee called for the establishment of an educational commission for health sciences . it also noted that medical school faculty , though efficient in their clinical specialties , were deficient as educators . it is commonplace to hear of faculty having been lured with exorbitant salaries or to hear of imposters being produced as faculty before inspecting teams from the medical council of india . much before the british stepped into india , residential universities in takshashila and nalanda provided organized institutionalized training in medicine . students were trained in both theoretical and practical aspects under the guidance of a guru or a preceptor . the unique feature of these universities is the presence of tutorial cells where a teacher took a fixed number of students under him / her and bestowed personal attention on them . student ratio in these gurukul systems closely corresponds to what is recommended by modern medical educators . in the blind race to produce a mass assembly line of medical graduates , that traditional concept of apprenticeship has been lost . only recently , there has been a resurgence of interest in faculty development and medical education . training and refresher courses in medical education have now been made mandatory for medical teachers . several efforts are being made to revamp the curriculum to make it relevant to the indian context . simultaneously , some efforts have been taken by the state to support the indian systems of medicine . in 1995 , this was renamed as the department of ayurveda , yoga and naturopathy , unani , siddha , and homeopathy in november 2003 . this was done to promote indigenous systems , upgrade their standards , ensure quality control and standardization of drugs , and improve educational standards and research in these areas . medical students who are taught in tertiary hospitals do not feel at home when they are asked to practice in rural areas . forced and compulsory postings to fulfill the dearth of medical personnel in rural areas are resented by fresh graduates . on the other hand , from colonial times , the state has been apathetic to the practitioners of indigenous medicine who serve rural populations . lack of societal respect and support forces them to practice allopathic methods , which is beyond their expertise , often with disastrous consequences . the focus of the indian systems of medicine has always been on health promotion and prevention , rather than the curative focus of the west . had a dialog been successfully initiated between these two systems , symbiotic growth might well have led to better health for our people . the abject lack of communication and learning between the western system of medicine on the one hand and indigenous systems of medicine on the other hand has not worked well for the country . medical education in india needs to transform to be meaningful for our own people . for this , the health agenda need to be accorded importance in governance . absence of a clear vision , a concerted effort by all stakeholders , and a strong political leadership in the area of medical education are the significant causes for the slow progress toward this metamorphosis . the prime task that lies ahead for india is to work out a national medical curriculum which caters to our country 's needs . it is also important to streamline the process of faculty development in the country and make it effective so that what is taught in workshops can be transferred into the workplace . the indigenous branches need to strengthen their research , improve the structure of their curricula , and standardize the regulation of their education system . eventually , a symbiotic relationship will have to be developed between the two systems of medicine , but before that , a lot of groundwork will need to be done in each system individually . merely mimicking the west , without paying heed to local priorities of course , there is always something that can be learned from any tradition , but it can only be to mutual benefit if done in a respectful manner . in the words of mahatma gandhi , i do not want my house to be walled in on all sides and my windows to be stuffed . i want the cultures of all the lands to be blown about my house as freely as possible .

published reports on salvage treatment for trachea reconstruction after total thyroidectomy or partial tracheotomy are available , some of them using structures of the trachea itself , auricular cartilage , a musculocutaneous flap , or other methods . in our report , we emphasize the importance of a search for a new material and approach for sparing surgery . the purpose of this article is to describe a case of a successful sparing surgery in a patient with advanced thyroid papillary carcinoma invading the trachea . after total thyroidectomy in 2012 , partial resection of the trachea was performed in 2014 . the lesion defect was 5.5 2.3 cm in size , located between 4 ( 2nd6th ) tracheal cartilaginous rings and involving about a semicircumference . it was reconstructed with the aid of the knitted tini - based mesh endograft , which has been prefabricated in the sternocleidomastoid muscle and further covered with the skin draped over the wound . the tracheostoma was fully closed 6 weeks after the surgery . there were neither side effects nor complications . this kind of tracheal surgery for extensive lesions demonstrates good functional and cosmetic outcomes . the cross - cultural exchanges between the people of india and their colonial rulers provides a fascinating insight into how these encounters shaped medicine and medical education in india . this article traces the history of how indian medicine was transformed in the backdrop of colonialism and hegemony . it goes on to show how six decades after independence , we have have still been unable to convincingly shrug off the colonial yoke . india needs to work out a national medical curriculum which caters to our country 's needs . a symbiotic relationship needs to be developed between the indigenous and allopathic systems of medicine .

in the recent past , even many historians of medicine and science have endorsed the widespread belief that the exodus of central european scientists and physicians during the nazi period could readily be described in terms of a linear equation of the subtractions and additions of intellect . this common interpretation has simplistically viewed the massive exodus of academics , intellectuals , and scientists after 1933 as an enrichment primarily of the north american and british medical and academic communities ( see medawar & pyke , 2001 or cornwell , 2003 , for example ) . although such a perspective is not entirely wrong when a rather quantitative meta - perspective is taken , it becomes less compelling when the individual biographies of the respective physicians and scientists themselves are taken into account and are placed in their contingent work environments . this includes their work situations , skill sets , along with the personal and psychological resources of each migr neuroscientist ( cf . this contribution introduces some of those local and cultural factors which implicated the arrival , acceptance , and integration of many german - speaking migrs doctors and brain researchers into canada , following their exile between 1933 and 1945 , which have largely gone unnoticed by the relevant scholarship on twentieth - century history of neuroscience . when tracing their career paths into the 1960s , during which the scientific research landscapes in canadian biomedicine gradually came to change with the creation of the medical research council ( mrc ) , the complex cultural modes and scientific interchanges associated with the forced migration process become fairly obvious ( mrc , 2000 ) . as the main title ( learning soft skills the hard way ) of this article implies , the integration of german - speaking migrs neuroscientists can not simply be perceived in terms of a supplementation of longstanding north american scientific traditions but needs to be viewed as a very complex process of acculturation on multiple levels of the social and cultural organization of contemporary canadian and american research landscapes . it is further more of a seemingly modern interest in the cultural makeup of science to analyze and understand the process of forced migration in the neuroscientific field while mapping the often drastic changes that took place to the career patterns of this particular group of medical professionals ( zeidman , 2014 ) . based on the existing historical evidence , the traditional views on the forced migration process in the neurosciences and psychiatry need to be significantly readjusted and refined . although most core facts about the exodus of medical researchers during the period of nazism in germany are already known ( cf . 191261 ) , a major incentive to revise some of the standard approaches stems from the historiographical problem of emigration - induced change , which has been researched from multiple perspectives in the humanities and social sciences . not only did scholars draw on individual and collective biographies but they also measured substantial impact parameters , using bibliometric methods , membership issues in academic associations , and statistics regarding the leading positions in scholarly societies , which were particularly applied to the hard sciences , such as physics and chemistry , as well as sociology and political science in the soft sciences ( juette , 1990 ; soellner , 1996 ) . the impulses for such a revised research strategy came from relatively new approaches to the historiography of the cultural context of science ( galison , graubard , & mendelsohn , 2001 ; schmidgen , geimer , & dierig , 2004 ; erikson , 2005 ) and problems of the transfer of knowledge ( argote , 1999 ; jankrift & steger , 2004 ; ash & soellner , 2006 ) . in applying those new perspectives to the research networks and the communication structures of migr neuroscientists , this article aims to provide additional perspectives towards the social background and cultural implications of the case of forced migration in the neuroscience field ( cf . an earlier process - oriented perspective developed in the 1990s by a group of scholars at the berliner wissenschaftskolleg has opened promising paths for the study of [ the ] intellectual and cultural change occurring through the forced migration of european scientific migrs ( ash & soellner , 1996 , pp . a number of american and german historians and philosophers of science ( fischer , 1996 ) have provided useful models through their scholarship on emigration - induced scientific change . these included the relevant social accounts of the historical developments , social reception , and reintegration of german - speaking migr scientists . as such , refugee - neuroscientists , like all their compatriots in exile , found themselves in the foreign environment of north america , where they had to continue their daily life , support their partners and families , become relicensed and obtain professional acceptance . they had to learn the social and cultural codes , psychological mentality , and likewise soft working skills 187197 ) when being barred from clinical work , having to close labs in order to pursue better paid jobs for their subsistence , or changing their personal research interests so as to fit more closely with the acceptable clinical and scientific paradigms of the often hands - on , capitalist , and technophile north american society . many of the migr neuroscientists , just to name the neuropathologist karl stern ( 19061975 ) , and his colleagues from the former group of kurt goldstein ( 18781965 ) , adhmar gelb ( 18871936 ) , victor franz ( 18831950 ) , and walther riese ( 18901976 ) in frankfurt am main , were influenced by interpretations of holistic neurology and the experimental culture of the weimar period , which they at first sought to continue in their american exile ( stahnisch & pow , 2015 ) . what emanates as the central problem for migr neuroscientists such as stern and goldstein , was not only personal acculturation but also the readjustment of their research and clinical activities . they had to search for new work places and integration into the preexisting canadian and us working groups , research programs , and academic milieus , which they often literally encountered as a new world . continuous comparisons of the similarities and differences with their former european experiences were permanently present ( sachs , 1998 ) , a process through which they noticeably stood out due to their critique and reproaches of the differences , shortfalls , and exaggerations of life in their new host countries . for example , the goldstein collaborator from berlin max bielschowsky ( 18691940 ) wrote back from his own exile abroad : i am as well as a man with my past could be in a very strange country [ auf fremdem boden ] . all the friendliness and kind offers of support by my [ new ] colleagues , however , will never really substitute for what i had to leave behind [ in germany ] . i am as well as a man with my past could be in a very strange country [ auf fremdem boden ] . you know how much i love my home country [ meine heimat ] . all the friendliness and kind offers of support by my [ new ] colleagues , however , will never really substitute for what i had to leave behind [ in germany ] . the members of the goldstein group certainly proved to be no exception to that rule , no matter what their influential contributions to neurology , psychiatry , experimental psychology , or matters of the philosophy of science and medicine had been . this loose network of people included the earlier collaborators from frankfurt am main and berlin , karl stern who had joined the allan memorial institute of psychiatric research at mcgill university in montreal , canada ) , walther riese who immigrated to richmond , virginia , in the united states , frieda fromm - reichmann ( 18891957 ) who received a position as a psychiatrist at the chestnut lodge mental asylum in maryland in the united states and who independently immigrated to other destinations in north america . goldstein s nearest friend and colleague adhmar gelb ( 18871936 ) had lost his chair at the university of halle and was just about to leave germany in 1936 for a position at kansas state university in manhattan , kansas , when he succumbed , at the age of 49 , to a tuberculosis infection , which he had contracted in his continuous work with severe clinical patients ( danzer , 2006 , p. 23f ) . the case of goldstein s collaborator stern in montreal , a former pupil of brain oncologist walther spielmeyer ( 18791935 ) in munich , can be presented here as an important change from an accomplished neuropathologist back in germany , who later became a well - accepted clinical psychiatrist and fervent academic teacher later in canada . at first glance , the conditions for a transfer of concepts and methods were ideal in stern s case ( goldblatt , 1992 , pp . 279282 ) , who was born in a small town in bavaria near the czech border ( bullemer , 2003 ) . karl - stern - strae in his honor and has commemorated his expulsion from germany . after he had passed most of his medical education at the charit hospital and medical school in berlin , stern graduated with an md in 1930 from the university of frankfurt / m . between 1930 and 1931 , he worked together with goldstein as a resident physician in psychiatry at the frankfurt neurological institute ( stahnisch , 2008 ) . karl - stern - strae in his honor and has commemorated his expulsion from germany . between 1932 and 1933 , he had a rockefeller fellowship in the department of neuropathology at the german research institute for psychiatry ( deutsche forschungsanstalt fuer psychiatrie : dfa ) in munich to collaborate with the neurohistologist spielmeyer , one of the world - leading specialists at the time for brain - tumor diagnoses which provided the basis for fruitful scientific publications ( stern , 1939 ) . here , he had procured a position , in which , apart from the pathological analysis of the brains in idiocy and circulatory disturbances , he mainly acted as spielmeyer s teaching assistant . yet , this implied an enormous effort to live up to the high standards of spielmeyer s expertise in this area . ( this is all the more crucial , as one of the members of the leading spanish school rafael lorente de no [ 19021990 ] and po del rio hortega [ 18821945 ] and oskar [ 18701959 ] and ccile vogt [ 18751962 ] in berlin he was also expected to introduce graduate students and visiting researchers into the various histological methods and the vast array of laboratory applications in use by spielmeyer so that for a large part of the day , stern had to wander from microscope to microscope in order to instruct the guests in munich , stern clearly worked at the cutting - edge of neuroscience research and medical education at large . when goldstein decided to leave frankfurt am main in 1930 for berlin ( kreft , 1997 , pp . 131144 ) , he asked stern to join him again as a consultant in one of his psychiatry wards . since stern had by that time received a great reputation for being a proficient neurohistologist himself , he was also expected to do the brain autopsies in the moabit prosectur . it seems that stern , with his broad interests and knowledge basis , squared very well with goldstein s holistic neurological assumptions which integrated philosophy , social psychiatry , and neuroscientific innovations alike . holist neurology sat at the center of what nazi ideology later rejected as weak jewish medicine , while stern himself did not hesitate at all to follow his mentor to the german capital : the hours were from six to eight but frequently we worked until well after midnight . there i found myself in a strange and extraordinary world , entirely different from anything i have ever seen before . there were mothers with children who had just left a home destroyed by an alcoholic . these were drunkards , morphine and cocaine addicts , the hopeless , the destitute , those who had cynically and rebelliously isolated themselves , bound to a life of increasing solitude and destruction , and those who had succumbed to the deficiency of a loveless world . it was that fringe of life where human existence is ultimately atomized and surrounds itself with a void , a space of negation . it would take a whole book to describe all this so that the reader would be able to re - experience it . [ that means that i never recovered the undergraduate s boundless admiration for science and for the absolute sacredness of research . [ ] although i had more scientific training later , i never forget those experiences in moabit . they seemed to have put the abstract scientific aspect of medicine into its proper place . it is just one side of a profound and complex development that with many of us science and art in medicine are no longer integrated . as science in general , ( stern , 1951 , p. 85f ) the hours were from six to eight but frequently we worked until well after midnight . there i found myself in a strange and extraordinary world , entirely different from anything i have ever seen before . there were mothers with children who had just left a home destroyed by an alcoholic . these were drunkards , morphine and cocaine addicts , the hopeless , the destitute , those who had cynically and rebelliously isolated themselves , bound to a life of increasing solitude and destruction , and those who had succumbed to the deficiency of a loveless world . it was that fringe of life where human existence is ultimately atomized and surrounds itself with a void , a space of negation . it would take a whole book to describe all this so that the reader would be able to re - experience it . [ that means that i never recovered the undergraduate s boundless admiration for science and for the absolute sacredness of research . [ ] although i had more scientific training later , i never forget those experiences in moabit . they seemed to have put the abstract scientific aspect of medicine into its proper place . it is just one side of a profound and complex development that with many of us science and art in medicine are no longer integrated . as science in general , ( stern , 1951 , p. 85f ) moabit was then one of the few academic hospitals with different services in neurology , psychiatry , and pathology that similarly related to each other as in the huge neurological institute , which goldstein directed in frankfurt before . however , just as everything was set for goldstein s clinic to develop into one of the major centers of german neurology , the catastrophe began . as soon as the nazis had seized power , goldstein was incarcerated and only released after agreeing to leave germany forever . through switzerland , where he cofounded the emergency society for german scholars in exile ( notgemeinschaft deutscher wissenschaftler i m ausland ) together with the budapest pathologist philip schwarz ( 18941962 ) and the mainz novelist carl zuckmayer ( 18961977 ) , he sought refuge in amsterdam , finishing his seminal publication deraufbau des organismus [ the architecture of the organism ] ( harrington , 1991 , pp . stern stayed in germany until 1935 , before he left for london and eventually reached new york . here a tight networking between contemporary international scientists comes into play , as his mentor from munich days , walther spielmeyer , had met the montreal neurosurgeon wilder penfield ( 18911976 ) , previously familiar with stern s work , on penfield s lecture tour to north and south america in 1931 ( weber , 2000 , p. 240f ) . also , stern s new acquaintance with a canadian neurophysiologist at queen square who had supposedly been herbert h. hyland ( 19001977 ) from toronto and who was in london exactly during this time helped likewise so that stern could leave for montreal , where he immediately began to work in a mental hospital then on the outskirts of the city ( hspital de ntre dame ) . spielmeyer had already gotten in contact with penfield through letter communication by otfrid foerster ( 18731941 ) . he was later invited by the montreal neurosurgeon to visit the neurological institute of mcgill university on his lecture tour to north america . also a transatlantic contact endured between goldstein and franz alexander ( 18911964 ) , since both had frequently been encountered at the joint seminars of the neurological institute with the psychoanalytical institute in frankfurt am main ( see laier , 1994 , pp . as penfield was to inaugurate a psychiatric department to complete his neurological institute , he recommended stern to the biological psychiatrist d. ewen cameron ( 19011967 ) as the designated director . shortly after the allan memorial institute ( ami ) had opened , in 1943 , stern was explicitly working for the latter s geriatric unit together with the czech - born and german - trained physician vojtech albert kral ( 19031988 ) ( see fig . 1 ) . he further taught the students courses as a research assistant , and later as an assistant professor for psychiatry ( hogan , 2007 , pp . however , as stern admits in his autobiography the pillar of fire , his interests in neuro - oncology and the cognitive defects in clinical psychiatry went further than the narrow program , as well as the the routine culture at the montreal neurological institute ( mni ) and the royal victoria hospital . in fact , stern came into a preexisting interdisciplinary hospital setting , which was soon conceptually and also locally separated between its main players , the mni and ami . the mni mainly fulfilled penfield s specific needs , that is , the different departments of epileptology , neurosurgery , neurology , and neuropathology were service institutions for an extended research program for the mapping of the human cortex . not regarding the personally problematic relationship between renowned professor cameron , and the migr psychiatrist stern on his staff , stern also left montreal at the end of the 1950s to assume a leading role in clinical psychiatry in ottawa . between 1951 and 1975 , he continued to work as a clinical psychiatrist at the university of ottawa , yet no longer being a laboratory brain researcher . for more than a decade , he also served as the head of the psychiatry department and promoted an integrative clinical approach that also encompassed psychoanalytical therapy options ( stahnisch & pow , 2015 , p. 246).figure 1 . karl stern ( first person right of d. ewan cameron in the center ) at the allan memorial institute ( ami ) , circa 1946 . theodore i. sourkes , mcgill university , montreal , canada . reproduced by permission of dr . spielmeyer had already gotten in contact with penfield through letter communication by otfrid foerster ( 18731941 ) . he was later invited by the montreal neurosurgeon to visit the neurological institute of mcgill university on his lecture tour to north america . also a transatlantic contact endured between goldstein and franz alexander ( 18911964 ) , since both had frequently been encountered at the joint seminars of the neurological institute with the psychoanalytical institute in frankfurt am main ( see laier , 1994 , pp . karl stern ( first person right of d. ewan cameron in the center ) at the allan memorial institute ( ami ) , circa 1946 . conditions for the transfer of ideas and methods were ideal in the case of karl stern , although his biography can not really be regarded as a success story in terms of theory change in the neurosciences . on the one hand , goldstein s group , to which he belonged in the early 1930s , was about to transfer moabit hospital into one of the country s major centers for neuroscientific research , but the machtergreifung of the nazis diminished all their plans . it represents in a nutshell many other areas of medical science that stood in opposition to the ideals of nazism and could not continue as traditions in germany . with a view to the cultural picture of science , holist neurology ceased to exist , when the goldstein group continued its work in spheres of clinical and experimental psychology . on the other hand , stern himself came into a preexisting interdisciplinary hospital setting at the mni , which was highly organized , although not in a broad and holistic fashion as many german centers . it rather fulfilled penfield s and later cameron s specific research needs ( feindel , 1991 , p. 821f ) . this story is far from complete , however , if it is not considered in terms of personal success and institutional change . numerous oral history accounts underline stern s noteworthy talent as an academic teacher , who seemed to have interested a whole new generation of montreal medical students in the histological study of the brain ( feindel , 1984 , pp . it also informs us about the necessity of broad education and training , often forgotten by a disciplinary tunnel vision on scientific excellence disrespecting a solid training base as the source for future innovations . the view on the cultural picture of science thus shows that stern s life and work was doubly prevented from blossoming into a full biomedical career in the beginning years by national socialist - politics and then as a coworker to cameron s program . his case can thus be seen as a conversion from a basic neuroscientific researcher into a fervent clinician and influential university teacher . thus , stern s case counts in favor of the assumption that emigration - induced scientific change must be separated from general scientific change at various levels from the individual to the cultural , although in this example this would have to be done in a narrow if not to say negative sense . in contrast with the first example and despite the emigration of a mind with its methods , no thorough induction of scientific change can be identified in stern s case , but contrafactually might have well been , if the facilities at moabit hospital had not been resolved by nazi officials . similar to stern s individual fate in canada , the goldstein group fell apart and the research of its members took on a very different direction . goldstein himself entered a private practice of neurology and psychiatry , after he had arrived in new york city in 1935 , dispersing his own work between an appointment as clinical teacher of psychopathology at columbia and further running a small neurophysiological laboratory at montefiore hospital . although he stayed in close letter exchanges with other diaspora members of this former group , they all now went their own ways , such as the neuropathologist walther riese ( stahnisch & pow , 2014 , pp . riese also left holist neurology and ventured into theoretical neuropsychology , and later medical history . goldstein s former clinical psychologist , adhmar gelb , had died in 1936 , after losing his chair at the university of halle and not living up to travel to the united states , where he was to assume a professorship in experimental psychology offered to him by kansas university . the decline of the school was further reflected in stern s fate in canada , who rather supplemented the neuropathological expertise at montreal s mni and ami , before deciding to continue his work later as a clinical psychiatrist in ottawa . university archives of the university of ottawa , on ( fonds 43 nb-3056 , karl stern , human resources files ; fonds 6 nb-9656.8 ) , passim . university archives of the university of ottawa , on ( fonds 43 nb-3056 , karl stern , human resources files ; fonds 6 nb-9656.8 ) , passim . with a view to the cultural perspective of scientific and clinical practice , it is certainly possible to see the cases of stern , goldstein , and riese not simply as additional biographies related to the forced migration wave from germany . instead , their histories tell us more about the actual production processes of knowledge in medicine and neuroscience . on the one hand , goldstein s group was clearly about to transfer moabit hospital into an important center for neuroscientific research in germany , but the machtergreifung of the nazis diminished all their plans . on the other hand , all of these migr neuroscientists came into preexisting clinical and research settings with their specific interplay of conceptual , personal , and research relations , in which , taking up a word of stern s ( 1951 , p. 77 ) , methods [ had already ] become mentalities . that is , they had to cope with the local north american research cultures and mostly had to abandon their own holistic ideas to more applied forms of neurology and patient testing , however , still influencing local practices : in stern s case , a strong emphasis on psychoanalytical psychopathology , in goldstein s example , a thorough way of clinical observation and history taking , and riese served as an important role model in his faculty , combining in - depth neuropathological knowledge with clinical alertness and a wide range of historical and interdisciplinary scholarship . so in their local settings , there still survived a bit of holist patina , which impressed faculty colleagues and strongly influenced their students . and stern was also very influential in his relation to the younger faculty members at the ami , for example , as the later psychiatrist and psycho - immunologist dr . edrita fried ( b. 1934 ) , associate in stern s service , has stated ( sourkes & pinard , 1995 , p. 151 ) . the instances of scientific and knowledge changes that can be extracted from this case example both apply to laboratory and clinical practice as well as to the emergence of new kinds of interdisciplinarity : the reconstruction of differing neuroscientific research styles or cultures hence shows the necessity to go beyond the more classical perspectives of the history of ideas , of institutional historiography , or the writing of individual scientists biographies and to take the communication and teaching networks of the migrs into account as well . this holds for the cultural patterns inscribed into thought and practice , national identities , and international contacts during the constitutional phase of the emerging neurosciences ( rosen , 1944 , p. 39 ) . the role of the stranger in creating innovative fields and disciplines in new cultural environments is of pivotal importance . just as the social need for comparison in the immigrant individual becomes a vital property for adaptation in the new cultural surrounding , the ability to criticize and relate to preexisting research traditions assumes ample input from local cultural values , readily shaped interpretations of new observations , or clinical skills . an important , though quite dated source in related medical historiography is rosen ( 1944 , p. 39 ) . thus , it becomes possible to study the interdisciplinary exchanges in a rather in - depth manner as these continued in both collaborative clinical and theoretical work despite the disrupted and dispersed local contexts along the american east coast , mediated by letter exchanges , phone calls , and the still very dense railway system in the 1950s . the role of the stranger in creating innovative fields and disciplines in new cultural environments is of pivotal importance . just as the social need for comparison in the immigrant individual becomes a vital property for adaptation in the new cultural surrounding , the ability to criticize and relate to preexisting research traditions assumes ample input from local cultural values , readily shaped interpretations of new observations , or clinical skills . an important , though quite dated source in related medical historiography is rosen ( 1944 , p. 39 ) . with respect to the north american medical context in the 1930s , and apart from the philanthropic endeavors of the emergency committee for displaced physicians ( zimmerman , 2006 ) , and the assumed responsibility of the rockefeller foundation for its former fellows and awardees , there had to be either a substantial need for research expansion or some perceived deficiency in scientific competences and clinical care , before the knowledge of the migrs could come into play ( pearle , 1984 ) . until that was the case and even in times of the transition into migrs resumption of professional work , they relied heavily on scientific colleagues , politicians , business men , and even family members to facilitate their reintegration process in their new host countries . in fact , landing in the united states outside of the contemporary population quotas for german , austrian , czech , and polish immigrants or without sufficient proof of having been a university teacher in the country of origin was only possible through the individual affidavit of american citizens who declared to sustain migrs in times of financial hardship ( davie & koenig , 1949 , pp . beyond migrs positive experiences , the failures , backlashes , and even hostilities that many of the migrs neuroscientists had to face in their private and working lives , deserve further scrutiny . this was particularly the case during the early years following their arrival in a generally anti - german and often even outright anti - semitic climate before the war , which led to their exclusion from the professional job market , cultural misunderstandings as to their former positions , along with insufficient language proficiency that created many disturbances among their academic peers ( stortz , 2003 , pp . in addition , there was also a widespread mood of resignation among many of the german - speaking migrs , particularly during the first three years of the war , when the blitzkrieg brought many european countries under nazi occupation and when family members had been imprisoned or even interned in penitentiaries and concentration camps . for some , they simply had not received any news from their loved ones on the other side of the atlantic . like many other contemporary immigrant groups , german - speaking migrs also used to stay together in similar neighborhoods of major north american cities , such as the lower east side in new york city , clayton neighborhood of st . their constant devaluations of american culture were proverbial , with ongoing exchanges about their former experiences from previous lives in central europe . in their kaffeekraentzchen , salons , and gespraechsrunden , which often became known as the group meetings of the beiunskys ( bien de chez nous ) ( sachs , 1998 ) , there was no separation by profession between scientists , artists , and writers , of course , serving the basic functions for moral and practical support in continued interdisciplinary exchanges ( grob , 1983 ) . by applying a network - oriented approach to such historical processes , the classical perspectives confined to certain types of gains and losses in differing neuroscientific research styles such a network approach may indeed be seen as a reformulation of what harvard historian of science thomas s. kuhn ( 19221996 ) had once called a disciplinary matrix ( kuhn , 1977 ) , that is , the commitment and involvement of individual scientists to the shared conceptual resources , values , instruments , techniques , and practices of their respective community . thus , in the field of the neurosciences , the actions of the main players and mediators of such a matrix may be feasibly reconstructed with regard to varying organizational and contextual points ( meyer , 2001 , p. 93 ) . regarding such specific scholarly networks in relation to others , it has to be born in mind that their forms and characteristics varied markedly due to their intrinsic composition through the academics , economists , politicians , and nonprofessional actors involved . however , their results may in the end be quite equivalent , as most academic , clinical , or organizational positions were acquired via personal relations , academic references , and the reputation of the teaching or research institutions at the time . this leads also back to the central question on the elements that have triggered and fostered the theory - change in the neurosciences under various cultural , social , and institutional conditions . exemplary are the official as well as unofficial networks within the german research society , the kaiser wilhelm society , and the early german research institution for psychiatry that played major roles in the support , placement , and connection of the migr neuroscientists from central europe in the united states and canada ( hammerstein , 2000 , pp . here , it is important to methodologically integrate the status of collective biographies , scientific networks , and interdisciplinary endeavors into this particular historical analysis of knowledge change in the neurosciences . in this respect , it will have to be kept in mind that not only highly skilled individual researchers had to leave central european universities during the nazi period , but also often whole research schools were forcefully expelled from the german - speaking countries . as an example , i want to draw the attention to the case of clinical neurologist and psychiatrist robert weil ( see fig . 2 ) , who belonged to a group of german - educated neuroscientists of the provinces of the former austrian - hungarian double - monarchy . he was born into a jewish family in a rural part of bohemia , yet in his adolescence he converted to lutheran protestantism . weil was one of many clinical and social psychiatrists during the 1930s , who displayed a profound research interest in various areas of psychiatry , ranging from nosology , psychoanalysis well over to the neuro- and histopathology of the brain . he had graduated from the german university of prague in 1933 and pursued postgraduate studies in neurology and psychiatry at the vienna medical school . he then worked as an army psychiatrist between 1935 and 1938 in prague and in bohemia , before he fled together with his family for the united kingdom and later to canada , following the annexation and the ceding of parts of czechoslovakia to germany ( baglole , 2002 , p. 64 ) . with many other migr medical scientists , after the passage to canada , weil shared the fate of many migrs of being transported to one of the more remote areas of saskatchewan in the prairies , where he was allowed to practice medicine as a general practitioner between the years of 1939 and 1942 . until 1944 he interned in neurosurgery at the saskatoon city hospital and eventually during the war years , weil managed to work with the saskatchewan mental health service until 1949 . predominantly practiced under poor conditions in mental hospitals , and in which he regarded university psychiatric teaching to be uncommon as a subject of study in canadian universities . letter of robert weil , halifax , ns , to the psychiatrist dr . charles a. roberts ( 19181996 ) in ottawa , dating june-7 , 1986 : in 1942 i joined the mental health services of saskatchewan , my first position [ in canada ] being a junior psychiatrist in the sask . clarence [ m. ] hincks [ 18851965 ] who visited our hospital almost yearly . on one of these visits he was accompanied by dr . both sat in on our conferences and also gave us the opportunity to meet and get to know them in personal conversations . both clarence and jack were always welcommed [ ! ] [ sic ] visitors who brought us all kind of informations [ ! ] [ sic ] about the psychiatric activities , developments etc from all the provinces . they also shared with us their visions and plans for the improvements in the care for the mentally ill . ; citation taken from his typographed letter from the folders on the robert weil correspondence ( ms 2 - 750 , call # 2003 - 47 , box 8 , file 15 , p. 1 ) in the dalhousie university archives & special collections , killam memorial library , halifax , ns . in comparison , neurology and psychiatry at the charles university of prague weil s alma mater had previously risen to international recognition under arnold pick ( 18511924 ) and ladislav hakovec ( 18661944 ) , who expanded compulsory university training in neuropsychiatry , psychopathology , and areas of social psychiatry to all medical graduates.figure 2 . robert weil ( a very rare portrait photograph , c. 1984 ) from the program invitation to his funeral ceremony in 2002 ( robert weil correspondence , ms 2 - 750 , call # 2003 - 47 , box 6 , file 1 ) . dalhousie university archives & special collections , killam memorial library , halifax , canada . reproduced by permission of dalhousie university archives & special collections , killam memorial library , halifax , canada . charles a. roberts ( 19181996 ) in ottawa , dating june-7 , 1986 : in 1942 i joined the mental health services of saskatchewan , my first position [ in canada ] being a junior psychiatrist in the sask . mental hospital in battleford . clarence [ m. ] hincks [ 18851965 ] who visited our hospital almost yearly . on one of these visits he was accompanied by dr . both sat in on our conferences and also gave us the opportunity to meet and get to know them in personal conversations . both clarence and jack were always welcommed [ ! ] [ sic ] visitors who brought us all kind of informations [ ! ] [ sic ] about the psychiatric activities , developments etc from all the provinces . they also shared with us their visions and plans for the improvements in the care for the mentally ill . ; citation taken from his typographed letter from the folders on the robert weil correspondence ( ms 2 - 750 , call # 2003 - 47 , box 8 , file 15 , p. 1 ) in the dalhousie university archives & special collections , killam memorial library , halifax , ns . robert weil ( a very rare portrait photograph , c. 1984 ) from the program invitation to his funeral ceremony in 2002 ( robert weil correspondence , ms 2 - 750 , call # 2003 - 47 , box 6 , file 1 ) . dalhousie university archives & special collections , killam memorial library , halifax , canada . reproduced by permission of dalhousie university archives & special collections , killam memorial library , halifax , canada . consequently , in this institution and later by his colleagues of the saskatchewan mental hospital at battleford , it was realized that weil was a broadly trained psychiatrist and neurologist , who had a lot of experiences in field psychiatry , due to his earlier appointment in the medical service of the czech army . his biography thus represents one of numerous examples , in which a neuroscientist arriving from central - europe found poor clinical and mental health conditions in north america in comparison with those he was acquainted with in the german - speaking context . for weil s perception of the canadian context of psychiatry and mental health see reports ; notes & articles on interdisciplinary research ; materials from the folders of the robert weil collection ( ms 2 - 750 , call # 2003 - 47 , accession report , box 2 , file 5 ; box 5 , file 3 ) in the dalhousie university archives & special collections , killam memorial library , halifax , ns . as weil perceived it , the subject of mental health was quite uncommon at canadian universities and not very well established as a serviceable system in the rural areas of the country . at this critical juncture of his career , his broad knowledge in psychiatry , his engagement in setting up a wider mental health system , and his social contacts with leading members of the saskatchewan health service and the canadian psychiatric community earned him such recognition that weil was hired as the first assistant professor of psychiatry by dalhousie university in nova scotia . he even managed to get this post against the stern reluctance of the officers of the medical society of nova scotia and their earlier policy , lasting between 1910 and 1930 , not to accept aliens and jewish - born doctors from other countries ( hincks , 1947 , pp . weil stayed at dalhousie university from 1950 to 1975 and then retired at the level of an associate professor . as a member of its core faculty , he exerted a strong influence on the hiring policy of that university and the organizational restructuring of its services in psychiatry , neurology , and neuropathology , in which he promoted a it is remarkable , and yet typical of many other migr neuroscientists , that weil displayed a thoroughly scientistic attitude to a variety of perspectives on psychiatry . although strongly influenced by experiencing his and his wife s expulsion from their home country as well as the general political events , which he academically discussed and heavily criticized in many of his papers and articles . he believed in a unifying and quite cathartic effect of science on its digression in ideology and technocracy . in a way , a general outlook of science and the humanist attitude in psychiatry served for him as residuum non destructum in times of personal despair and general political worries after his immigration to canada : this discrepancy between our knowledge and our behavior makes it so difficult today to orient oneself in this chaos of our enigmatic world . man now stands confused before his own creation complicated by so many technical devices which he is unable to control . and in his confusion and his insecurity he is always more tempted to reach for a gun than for an instrument of peace . war appears to be still a better and safer alternative to a peaceful adjustment of our environment to our needs and a better adaptation of mankind to the material world which presents itself to us.as you see , there are two main problems we have to face in modern time . firstly , we have to utilize our knowledge for practical application that is a matter of economics and politics and therefore outside of my realm.the second large problem of mankind is a psychological one . it is the problem of the adjustment of individuals and groups to the environment which is set for him the moment he is born . it is also the problem of redirecting our mental potentialities to a healthier attitude towards the material world which surrounds us & towards our fellowmen . from weil s address to the cpa in 1953 ; citation taken from his manuscript typography from the folders on the robert weil correspondence ( ms 2 - 750 , call # 2003 - 47 , box 6 , file 15 ) in the dalhousie university archives & special collections , killam memorial library , halifax , ns . for weil s perception of the canadian context of psychiatry and mental health see group for the advancement of psychiatry . reports ; notes & articles on interdisciplinary research ; materials from the folders of the robert weil collection ( ms 2 - 750 , call # 2003 - 47 , accession report , box 2 , file 5 ; box 5 , file 3 ) in the dalhousie university archives & special collections , killam memorial library , halifax , ns . this discrepancy between our knowledge and our behavior makes it so difficult today to orient oneself in this chaos of our enigmatic world . man now stands confused before his own creation complicated by so many technical devices which he is unable to control . and in his confusion and his insecurity he is always more tempted to reach for a gun than for an instrument of peace . war appears to be still a better and safer alternative to a peaceful adjustment of our environment to our needs and a better adaptation of mankind to the material world which presents itself to us . as you see , there are two main problems we have to face in modern time . firstly , we have to utilize our knowledge for practical application that is a matter of economics and politics and therefore outside of my realm . it is the problem of the adjustment of individuals and groups to the environment which is set for him the moment he is born . it is also the problem of redirecting our mental potentialities to a healthier attitude towards the material world which surrounds us & towards our fellowmen . from weil s address to the cpa in 1953 ; citation taken from his manuscript typography from the folders on the robert weil correspondence ( ms 2 - 750 , call # 2003 - 47 , box 6 , file 15 ) in the dalhousie university archives & special collections , killam memorial library , halifax , ns . from weil s address to the cpa in 1953 ; citation taken from his manuscript typography from the folders on the robert weil correspondence ( ms 2 - 750 , call # 2003 - 47 , box 6 , file 15 ) in the dalhousie university archives & special collections , killam memorial library , halifax , ns . his work thoroughly introduced german ideas of social psychiatry and interdisciplinary teaching and research , while helping to establish a more effective level of education and patient care in the canadian public mental health system ( dowbiggin , 2003 ) . his stronger engagement with mental health issues can be seen as an individual example of change from his primary interest in somatic neurology and neuropathology . neither in the czech army nor in the canadian health system could his interests be fully met ( dalhousie university , 2002 , p. 1 ) . what this example shows , however , is definitely not that canadian social psychiatry and the development of dalhousie s facilities in the neurosciences could not have emerged without robert weil . it makes plausible that under his supervision and tutelage , the historical course taken would have had a different velocity and would have ventured into new directions ( weil & demay , 1947 ; weil , 1960 ) . the course of these events certainly appears to be a mixture of institutional circumstances and biographical factors , as this highly intellectual man , who not only reflected in numerous sociological and philosophical articles on the cultural background of the neurosciences but also shaped and reshaped the research outlook of areas of biological psychiatry and bench neuroscience at his university , proved himself to be an effective and pragmatic science organizer , who integrated these ideas into the institutional setting of his medical school . dalhousie , at that time , experienced an increase from two to eight professorships in neurology , psychiatry , neuropathology , and neuroanatomy , while he was an active faculty member . elective programme ; materials from the folders of the robert weil collection ( ms 2 - 750 , call # 2003 - 47 , accession report , 1 p. , box 5 , file 3 ; box 6 , file 11 ) in the dalhousie university archives & special collections , killam memorial library , halifax , ns . elective programme ; materials from the folders of the robert weil collection ( ms 2 - 750 , call # 2003 - 47 , accession report , 1 p. , box 5 , file 3 ; box 6 , file 11 ) in the dalhousie university archives & special collections , killam memorial library , halifax , ns . the central points addressed by weil also strongly influenced a development in canada , which gerald grob has characterized as the emphasis on the prevention and on the provision of care and treatment in the community for us psychiatry ( grob , 1983 , p. 232 ) . the mental health problem from large hospitalization numbers was perceived as highly demanding also by the psychiatrists of the canadian and american psychiatric clinics and asylums thus bearing widespread implications for the mental health system . it is important to see that migr doctors exerted a strong impact on the canadian and us mental health systems , which was , however , preceded by reevaluations going on in various parts of europe , mainly in areas of the former austro - hungarian empire , in italy , switzerland , and germany . these particular refugee scientists and medical doctors must have been well trained to arrive at a tenacity of solving problems and overcoming all sorts of constraints and obstacles in their clinical or laboratory research , as was the case with many migr neuroscientists , who had been trained in some of the leading centers in central europe . from a social history viewpoint , the emigration of scientists and scholars after 1933 can even be understood as a spectacular case of forced international elite - circulation , but that circulation did not happen automatically . instead , before we may take scientific change into account , we need to question who got the opportunity to continue or begin scientific work , and thus at least a potential position to participate in changes of research trends in his or her host country ? very likely , those individuals had to have the necessary aptitude to convince greater audiences , as well as the social and basic linguistic competences to negotiate budgets with administrative officers . that the requirement of such soft skills is far from trivial would become fairly obvious in the case of many migr neuroscientists , who often needed to pass medical exams before being allowed to practice again . they had to find jobs in research institutes or medical faculties , and were to serve in low - paid or nonsalaried voluntary positions , etc . for example , other research import products from migr neurologists and psychiatrists such as the neurorehabilitative approach of the holist neurologist kurt goldstein from frankfurt and berlin , the psychiatric genetics and epidemiology of the berlin psychiatrist franz josef kallmann ( 18971965 ) , or the introduction of the psychopharmacological chlorpromazine therapy chlorpromazine therapy had been introduced by the psychiatrist and biochemist heinz lehmann , who had promoted the development in the french - speaking literature also among the english - speaking north american research communities in psychiatry and neurology.for a long time stood in the cultural shadow of dominant psychoanalytical theories . in american psychiatry , the clinical psychoanalysts influenced both the state hospital system as well as the large psychiatric hospitals of the veterans administration between the 1940s and the 1960s , before these major approaches further developed into some important research traditions in the field of modern neuroscience . personal interview with mni neurologist fred andermann , may 26 , 2007 , in the faculty club of mcgill university in montreal , pq . moreover , heinz lehmann at the allan memorial institute , the psychiatry department at mcgill , contributed the first research publications on chlorpromazine in english in 1953 and , three years later , on the antidepressant imipramine ( anonymous , 1993 , pp . lehmann represented one of those german - speaking migr physicians with whom weil upheld continuous letter exchanges after their mutual emigration to canada . for example , letter on august 19 , 1953 by robert weil to heinz edgar lehmann about the issue of clinical hypnosis and their mutual work on the canadian special commission on hypnosis and hypnoanalysis . materials from the folders of the robert weil collection ( ms 2 - 750 , call # 2003 - 47 , accession report , box 2 , file 5 ; box 5 , file 3 ) in the dalhousie university archives . chlorpromazine therapy had been introduced by the psychiatrist and biochemist heinz lehmann , who had promoted the development in the french - speaking literature also among the english - speaking north american research communities in psychiatry and neurology . personal interview with mni neurologist fred andermann , may 26 , 2007 , in the faculty club of mcgill university in montreal , pq . for example , letter on august 19 , 1953 by robert weil to heinz edgar lehmann about the issue of clinical hypnosis and their mutual work on the canadian special commission on hypnosis and hypnoanalysis . materials from the folders of the robert weil collection ( ms 2 - 750 , call # 2003 - 47 , accession report , box 2 , file 5 ; box 5 , file 3 ) in the dalhousie university archives . when looking more closely at the contributions of individual migr neuroscientists , such as heinz lehmann at mcgill university , the role and influence of the process of reintegration of the exiled neuroscientists in canada and the united states becomes more comprehensible . the vital function served by north america was that of a safe haven for the expelled scientists and intellectuals , a harbor for ideas , epistemologies , and innovative experiments , and a refuge for europe s cast - off intelligentsia during the rise of the nazi tyranny , holocaust , and second world war . despite a certain amount of attention paid to these homeless intellectuals in recent publications ( weindling , 1989 ; deichmann , 1996 ; israel , 2004 ) , their impact on and relation to american science and medical culture has not yet been fully explored . for many , coming to north america was like : parachuting from europe into the new world of north american psychiatry at the very brink of wwii with nothing in my backpack other than kraepelin s and bleuler s guides to the diagnosis of the major psychoses , manic - depressive disorder , and schizophrenia . we had only two theories to explain the rest of the psychiatric illnesses , the neuroses and personality disorders : freud s psychoanalysis and pavlov s and skinner s findings on conditioning and learning . personal file ( professor h. lehmann ) , collections of the university archives , mcgill university , montreal , canada . parachuting from europe into the new world of north american psychiatry at the very brink of wwii with nothing in my backpack other than kraepelin s and bleuler s guides to the diagnosis of the major psychoses , manic - depressive disorder , and schizophrenia . we had only two theories to explain the rest of the psychiatric illnesses , the neuroses and personality disorders : freud s psychoanalysis and pavlov s and skinner s findings on conditioning and learning . personal file ( professor h. lehmann ) , collections of the university archives , mcgill university , montreal , canada . personal file ( professor h. lehmann ) , collections of the university archives , mcgill university , montreal , canada . faced not only with expulsion from academic working circles but also prohibited from pursuing their career ( a berufsverbot ) , many of the neuroscientists looked at , here , searched to escape the situation in the german - speaking countries and to establish a new professional life elsewhere . this was also the case in the clinical neurologist and psychiatrist lehmann , who had been born as the son of a jewish physician in berlin ( see fig . he had himself studied medicine in marburg , as well as at the psychiatric centers of freiburg in germany and vienna in austria . between 1935 and 1937 , he pursued postgraduate research while being a staff - attending physician ( assistenzarzt ) at the martin luther stift and at the jewish hospital of berlin . in 1937 , after he had been barred to continue his medical work as a hospital physician even for his jewish patients , he managed to immigrate to canada on a tourist visa . immediately after his arrival in montreal in quebec , he was offered the position of a hospital physician at the verdun protestant hospital , which became the main clinical center of the psychiatric research divisions of mcgill university s allan memorial institute ( ami ) . rising fast through the academic ranks , in 1947 , he even became its clinical chief , which was rather a parallel reflection of the research interests of the somatic psychiatrist cameron at the ami . yet , other than in the example of karl stern , lehmann could benefit strongly from the support of the scientific and clinical milieu of the ami with its contemporary biological research programs ( sourkes & pinard , 1995 , pp . at the same time , lehmann managed to turn his solid education in french language from his berlin high school times into a large medical asset ( lehmann , 1983 , pp . 145154 ) . shortly after chlorpromazine had been developed by the psychiatrists and neurochemists , jean delay ( 19071987 ) and pierre deniker ( 19171998 ) in france in 1952 after world war ii , lehmann introduced the drug among the english - speaking clinical neuroscientists in north america lehmann now redirected his own psychiatric research solely from a psychopharmaceutical perspective and particularly towards the treatment of schizophrenic patients . one could even go so far as to view his work at mcgill university as a stepping stone for his additional activities in psychiatry and public mental health in the provincial comit de la sant mentale de quebc , as an american fellow of the collegium internationale neuro - psychopharmacologium , along with being a canadian representative and expert for the world health organization in new york.figure 3 . osler library of the history of medicine , mcgill university , montreal , quebec , canada . reproduced by permission of the osler library of the history of medicine , mcgill university , montreal , quebec , canada . osler library of the history of medicine , mcgill university , montreal , quebec , canada . reproduced by permission of the osler library of the history of medicine , mcgill university , montreal , quebec , canada . a defining feature for the biographical differences between stern and lehmann had openly been their diverging socialization in the medical research landscape in germany before their forced migration to canada . of course , they both had excellent language proficiency in french , which they could further perfect while working in the french - canadian surroundings of montreal . however , both migr neuroscientists used these soft skills in quite different ways : while stern primarily related to the francophone scientific community of quebec and aligned through his scientific connections particularly with the psychoanalytically schooled psychiatrists of france and quebec , lehmann emerged as a decisive bridge - builder between the new biological tradition of psychopharmacology in france and french - speaking switzerland with their developed pharmaceutical and chemical industries . lehmann was an important gatekeeper , who could introduce these impulses and initiatives into the english - speaking world of north american psychiatry ( stip , 2015 , p. s5 ) . another instructive example of a successful , though slightly changing career in the clinical neurosciences , is the professional silberberg - couple from breslau , where the female pathologist ruth silberberg ( 19061997 ) had to work four years without salary while her husband , the neurohistologist martin silberberg ( 18951969 ) , had to change from one low - paid and short - term position to the next one , first working in halifax , nova scotia , in canada , then in new york city and st . louis in the united states . in his letter exchange with his mentor , the american physician leo loeb ( 18691959 ) , martin silberberg respectively wrote on august 4 , 1938 , on the occasion of a job opening at the middlesex university in massachusetts : dear dr . loeb , i am sick and tired of moving around , unless it means a definitive step forward . letter of martin silberberg ( st . louis ) to leo loeb ( staying at woodshole ) on april-4 , 1938 ; archives and rare books collection of the becker library , washington university school of medicine ( fc0002 , leo loeb , correspondence r - s , box 5 , folder : silberberg , martin and ruth ) . i am sick and tired of moving around , unless it means a definitive step forward . herefore [ ! ] letter of martin silberberg ( st . louis ) to leo loeb ( staying at woodshole ) on april-4 , 1938 ; archives and rare books collection of the becker library , washington university school of medicine ( fc0002 , leo loeb , correspondence r - s , box 5 , folder : silberberg , martin and ruth ) . letter of martin silberberg ( st . louis ) to leo loeb ( staying at woodshole ) on april-4 , 1938 ; archives and rare books collection of the becker library , washington university school of medicine ( fc0002 , leo loeb , correspondence r - s , box 5 , folder : silberberg , martin and ruth ) . and also the move of the sternbergs to the rockefeller institute in new york did not prove to be a great relaxation of their tense living circumstances : dear doctor loeb , nothing has been heard of promotion or raises of salaries , since dr . [ it is the policy of the school to exploit everybody and to make use of everybody s plight . the school has the highest percentage of jewish [ ! ] students , who are glad to pay fees that are about 30% higher than yale s or harvard s . on the other hand , the salaries paid to the faculty are ridiculous . but , what can we do , if the difficulties to obtain a fairly decent position are unsurmountable [ ! ] ? [ william c. ] von glahn [ 19001961 ] lets us have our own ways in research . letter of martin silberberg ( nyc ) to leo loeb ( st . louis ) on dec-2 , 1943 ; archives and rare books collection of the becker library , washington university school of medicine ( fc0002 , leo loeb , correspondence r - s , box 5 , folder : silberberg , martin and ruth ) . it is the policy of the school to exploit everybody and to make use of everybody s plight . students , who are glad to pay fees that are about 30% higher than yale s or harvard s . on the other hand , but , what can we do , if the difficulties to obtain a fairly decent position are unsurmountable [ ! ] ? [ william c. ] von glahn [ 19001961 ] lets us have our own ways in research . letter of martin silberberg ( nyc ) to leo loeb ( st . louis ) on dec-2 , 1943 ; archives and rare books collection of the becker library , washington university school of medicine ( fc0002 , leo loeb , correspondence r - s , box 5 , folder : silberberg , martin and ruth ) . letter of martin silberberg ( nyc ) to leo loeb ( st . louis ) on dec-2 , 1943 ; archives and rare books collection of the becker library , washington university school of medicine ( fc0002 , leo loeb , correspondence r - s , box 5 , folder : silberberg , martin and ruth ) . this is only one out of many examples that serves to illustrate that the north american context was bursting with all kinds of pragmatic problems , which the newly arriving migr neuroscientists had to master before they could resume their research and clinical work . originality in their scientific work certainly was an important factor to enter major research groups and to gain acceptance in the scientific communities in canada and the united states . nevertheless , innovative ideas and the mastering of methods , which were not then accepted in their new host countries , proved to be an ambiguous advantage for the migrs researchers . an abundant amount of methodological originality , and thus difference in clinical or research style , could easily lead to incommensurable scientific views to those held by the local research community ( as in the cases of holist neurology , the shock therapies in brain psychiatry , or the use of the new psychopharmaceutical drugs , which had yet not been introduced in north america ) . this made it even harder to find good integration into day - to - day - research work in normal science and implies that some narratives will clearly fall into areas of traditional science studies and historical epistemology , as they are current since the works of thomas s. kuhn ( 1962 ) , georges canguilhem ( 1966 ) , robert k. merton ( 1973 ) . this made it even harder to find good integration into day - to - day - research work in normal science and implies that some narratives will clearly fall into areas of traditional science studies and historical epistemology , as they are current since the works of thomas s. kuhn ( 1962 ) , georges canguilhem ( 1966 ) , robert k. merton ( 1973 ) . important in this context is that discussions on migr scientists and physicians had long centered on a preselected group of outstanding individuals , whereas less attention was paid to those more marginal in their field . it is therefore of great importance for further advancement in the historiography of forced migration to draw specific attention to such rather hidden biographies of normal scientists and to cases of unsuccessful adaptation in their specific contexts . this will serve the development of a better understanding with regard of the broad transformations and the knowledge transfer in the field of neuroscience . the application of such a perspective will further enable us to also answer such questions as to why it was that the mental health system in canada and parts of the united states appeared so underdeveloped in comparison with the contemporary state of psychiatry and public health in central europe . what were the factors that made new research initiatives possible and applicable in the north american contexts ? and which factors enabled german - speaking migrs in particular to overcome everyday problems , research constraints , and cultural differences to contribute to the research traditions of brain psychiatry and clinical neuroscience ? although this had not been an attractive situation , it was possible to find one individual among the migrs neuroscientists . an extraordinary pathologist ruth silberberg , a breslau - trained developmental brain scientist , who fled with her husband , the neurohistologist martin silberberg , first to halifax and eventually settled in st . ruth accepted invitations through the german pathological society and individual university institutes to give guest lectures and seminars in germany during the late 1950s and early 1960s . it was only at the time of martin s death that she decided to accept an adjunct professorship at the university of zurich where she frequently taught during the summer break . see in archives and rare books collection of the becker library , washington university school of medicine ( fc0002 , leo loeb , correspondence r - s , box 5 , folder : silberberg , martin and ruth ) , no page number . ruth silberberg intriguingly represents an migr researcher who had an important voice in both medical communities one that was strongly heard in her own field of pathology as well as in clinical education ( cf . see in archives and rare books collection of the becker library , washington university school of medicine ( fc0002 , leo loeb , correspondence r - s , box 5 , folder : silberberg , martin and ruth ) , no page number . as mentioned in the introduction of this article , a more or less unquestioned belief in the historiography of science and medicine at large suggests that the process of forced migration in twentieth - century medicine and natural science can be specifically viewed as a process leading to a brain gain of the receiving countries ( such as the united states and canada in north america ) ( quirke & gaudillre , 2008 , p. 442 ) . the related view has nearly gone uncontested , namely that it made no difference for a biomedical researcher to substitute frankfurt am main for ottawa , ontario , breslau for halifax , nova scotia ; or berlin for montreal , quebec , as the respective sites for their research programs and clinical activities . this assumption had some plausibility when compared with the careers of migr professionals in the arts , in politics , or in the legal sphere ( strauss & roeder , 1983 ) . at a second glance , however , the above position of international universality is also not compelling when it is compared with other immigrant groups in the arts and film actors of the hollywood entertainment industry . in these seemingly unrelated fields , a transfer of knowledge and people could not take place without having to face greater cultural problems ( taylor , 1983 , pp . the historical analysis of the group of migr neuroscientists thus presents itself as a most interesting test case for newer approaches in the historiography of science that have interpreted the evolution and aberrations of the biomedical enterprise on grounds of their entanglement with culture . using historiography as a detailed description of research , laboratory practices , and clinical care approaches allows for a more adequate view of the underlying historical processes , particularly an integration of various communities of neurologists , psychiatrists , and brain investigators into preexisting american research cultures . in order to come to terms with the cultural differences , which german - speaking migr neuroscientists experienced when they adapted to north american research and clinical institutions , scientific foundations , and structure of politically influenced forms of research organization , their experiences in the biomedical field at the end of the weimar republic and the beginning of the nazi period in germany 7387 ) . when considering the transfer of such multifaceted patterns of clinical and basic research , laboratory practices , and interdisciplinary linkages with mental asylums , as well as anthropological research institutions , especially the more hidden biographies and local research cultures in the context of the forced migration wave of german - speaking neuroscientists to canada and the united states , left numerous traces of the setbacks and challenges , which they encountered when they tried to recommence their careers in the north american scientific and clinical milieus . with respect to the case examples discussed in this article , we have seen that the appropriation of new laboratory practices and clinical concepts in the research communities also supported new forms of interdisciplinarity working relations that became so decisive for the neurosciences today . nevertheless , when looking at the personal experiences , group mentalities , and even the soft skills learned the hard way , it has likewise become tangible how the research programs of migr neuroscientists reflected their foregoing experiences in the medical and health care cultures from the late wilhelminian empire to the onset of nazism and fascism in central europe , as well as the problems and setbacks they encountered , when making the very demanding transition to north america and its preexisting research cultures . in total , 196 male and female libyan citizens from many governmental and private dental clinics in tripoli participated in the study . the participants were examined between january and march 2012 , and all of them gave informed consent for participation . the study comprised patients and control participants between 25 and 65 years of age who were divided into : group one ( 2535 years ) , group two ( 3645 year ) , group three ( 4655 years ) , and group four ( 5665 years ) . the diagnosis of periodontal disease was established according to the clinical parameters of the periodontal disease index ( pdi ) , also known as ramfjord index . for the pdi assessment , six teeth were evaluated : the upper left central , first premolar and right first molar ; and the lower right central , first premolar , and left first molar were measured . examination method was applied by using university of michigan 0 probe with william 's markings at 1 , 2 , 3 , 5 , 7 , 8 , 9 , and 10 mm . each sextant was designed as either healthy ( score 0 ) ; mild to moderate gingivitis , but changes not extending all around the tooth ( score 1 ) ; mild to moderate gingivitis extending all around the tooth ( score 2 ) ; severe gingivitis ( marked redness , bleeding tendency , and ulceration ) ( score 3 ) ; a probing depth less than 3 mm ( score 4 ) ; a pocket depth from 3 to 6 mm ( score 5 ) ; or a pocket depth exceeding 6 mm ( score 6 ) , according to the scores recorded at the index teeth . cp was defined as oral health status with a pdi score of 5 and 6 . oral health status with pdi scores between 0 and 4 genomic dna was extracted from buccal swabs using the protocol of isohelix buccal dna isolation kit , which contained solution ls ( lysis buffer ) , solution pk ( proteinase k ) 1 ml , solution ct ( capture buffer ) , and solution te ( tris - edta ) , ( rehydration buffer ) ( 24 ) . agarose gel electrophoresis was applied to examine the integrity of the genomic dna ( 25 ) . about 0.75 m of each forward and reverse primers were added to a reaction mixture containing 1x ( 1.5 mm mgcl2)2 gotaq reaction buffer , pcr nucleotide mix 0.2 mm each dntp , 1.25 l of gotaq dna polymerase ( 5 u/l ) , and nuclease - free water to final volume 50 l . the pcr program was performed for 35 cycles and consisted of an initial denaturation for 2 min at 95c , 35 sec at 95c for denaturation , 52c for 35 sec for annealing , and 72c for 35 sec for extension , followed by a final extension for 7 min at 72c ( 26 ) . agarose gel electrophoresis was applied to the pcr products ( 25 ) , then pcr products were purified using the protocol of the qiaquick purification kit to remove excess salts , pcr primers , and dntps ( 27 ) . main characteristics of vdr gene polymorphisms and primers sequencesa from naito et al . after purification of the pcr products , dna cycle sequencing was performed using of bigdye terminator v1.1 cycle sequencing protocol ; 0.75 m of forward apai and bsmi , and 0.75 m of reverse foki primers were prepared . in the reaction mixture , 6.6 l of nuclease - free water was placed , followed by 4 l of ready reaction premix , 2 l of bigdye terminator v1.1 , sequencing buffer ( 5x ) and 6.4 l of apai , bsmi , and foki . the pcr program was performed for 25 cycles and consisted of an initial denaturation for 1 min at 96c , 10 sec at 96c for denaturation , 50c for 5 sec for annealing , and 60c for 4 min for extension . sanger sequencing method was applied for capillary electrophoresis ; the run was performed on applied biosystem ( 3500xl genetic analyzer ) . all statistical analyses were performed using the spss for windows , version 15.0 ( spss inc . , frequency differences of apai , bsmi , and foki polymorphisms and departure from hardy weinberg equilibrium were tested by pearson 's chi - square test which was also used to quantify the association between cp and these polymorphisms . the level of significance ( p value ) less than 0.05 was considered statistically significant for all analyses . in total , 196 male and female libyan citizens from many governmental and private dental clinics in tripoli participated in the study . the participants were examined between january and march 2012 , and all of them gave informed consent for participation . the study comprised patients and control participants between 25 and 65 years of age who were divided into : group one ( 2535 years ) , group two ( 3645 year ) , group three ( 4655 years ) , and group four ( 5665 years ) . the diagnosis of periodontal disease was established according to the clinical parameters of the periodontal disease index ( pdi ) , also known as ramfjord index . for the pdi assessment , six teeth were evaluated : the upper left central , first premolar and right first molar ; and the lower right central , first premolar , and left first molar were measured . examination method was applied by using university of michigan 0 probe with william 's markings at 1 , 2 , 3 , 5 , 7 , 8 , 9 , and 10 mm . each sextant was designed as either healthy ( score 0 ) ; mild to moderate gingivitis , but changes not extending all around the tooth ( score 1 ) ; mild to moderate gingivitis extending all around the tooth ( score 2 ) ; severe gingivitis ( marked redness , bleeding tendency , and ulceration ) ( score 3 ) ; a probing depth less than 3 mm ( score 4 ) ; a pocket depth from 3 to 6 mm ( score 5 ) ; or a pocket depth exceeding 6 mm ( score 6 ) , according to the scores recorded at the index teeth . cp was defined as oral health status with a pdi score of 5 and 6 . oral health status with pdi scores between 0 and 4 genomic dna was extracted from buccal swabs using the protocol of isohelix buccal dna isolation kit , which contained solution ls ( lysis buffer ) , solution pk ( proteinase k ) 1 ml , solution ct ( capture buffer ) , and solution te ( tris - edta ) , ( rehydration buffer ) ( 24 ) . agarose gel electrophoresis was applied to examine the integrity of the genomic dna ( 25 ) . about 0.75 m of each forward and reverse primers were added to a reaction mixture containing 1x ( 1.5 mm mgcl2)2 gotaq reaction buffer , pcr nucleotide mix 0.2 mm each dntp , 1.25 l of gotaq dna polymerase ( 5 u/l ) , and nuclease - free water to final volume 50 l . the pcr program was performed for 35 cycles and consisted of an initial denaturation for 2 min at 95c , 35 sec at 95c for denaturation , 52c for 35 sec for annealing , and 72c for 35 sec for extension , followed by a final extension for 7 min at 72c ( 26 ) . agarose gel electrophoresis was applied to the pcr products ( 25 ) , then pcr products were purified using the protocol of the qiaquick purification kit to remove excess salts , pcr primers , and dntps ( 27 ) . after purification of the pcr products , dna cycle sequencing was performed using of bigdye terminator v1.1 cycle sequencing protocol ; 0.75 m of forward apai and bsmi , and 0.75 m of reverse foki primers were prepared . in the reaction mixture , 6.6 l of nuclease - free water was placed , followed by 4 l of ready reaction premix , 2 l of bigdye terminator v1.1 , sequencing buffer ( 5x ) and 6.4 l of apai , bsmi , and foki . the pcr program was performed for 25 cycles and consisted of an initial denaturation for 1 min at 96c , 10 sec at 96c for denaturation , 50c for 5 sec for annealing , and 60c for 4 min for extension . sanger sequencing method was applied for capillary electrophoresis ; the run was performed on applied biosystem ( 3500xl genetic analyzer ) . all statistical analyses were performed using the spss for windows , version 15.0 ( spss inc . , frequency differences of apai , bsmi , and foki polymorphisms and departure from hardy weinberg equilibrium were tested by pearson 's chi - square test which was also used to quantify the association between cp and these polymorphisms . the level of significance ( p value ) less than 0.05 was considered statistically significant for all analyses . the distribution of the participants according to age groups between group one ( 2535 years ) and group four ( 5665 years ) was 15.5 , 23.7 , 30.9 , and 29.9% , respectively . according to pdi , 99 participants ( 50.0% ) were diagnosed with cp and 97 participants ( 49.5% ) were diagnosed as healthy controls . the distribution of the pdi scores between 0 and 6 was 1.0 , 2.6 , 4.6 , 11.2 , 26.5 , 32.7 , and 21.4% , respectively . cross - tabulation statistical test , which is based on pearson 's chi - square test , was used to analyze the frequency distribution , in order to find relationship between cp and both sex and age , as well as to find an association between cp and apai , bsmi , and foki vdr snps . statistically significant association between cp and both sex ( p=0.000 ) and age ( p=0.001 ) was detected ( table 2 ) . association between chronic periodontitis and age and sex the level of significance ( p<0.05 ) . the flanking regions by apai , bsmi , and foki primers were analyzed ; the lengths of the pcr products were 156 , 106 , and 146 bases , respectively . figure 1a shows a part of the sequence of the region flanked by apai primer including g / t snp rs#7975232 . in this study , the variant , c / t rs#731236 snp was detected in the region flanked by apai primers ( fig . figure 1c shows part of the sequence of the region flanked by bsmi primers including a / g snp rs#1544410 . figure 1d shows part of the sequence of the region flanked by foki primers including a / g snp rs#2228570 . ( a ) apai g / t snp ( highlighted in black , ambiguity code : k ) . ( b ) apai c / t snp ( highlighted in black , ambiguity code : y ) . ( c ) bsmi a / g snp ( highlighted in black , ambiguity code : r ) . ( d ) foki a / g snp ( highlighted in black , ambiguity code : r ) . the analysis of genotypic and allelic frequencies of the analyzed polymorphisms showed that a significant association p = 0.022 and p=0.023 , respectively , was foundbetween cp and apai c / t snp rs#731236 , whereas no statistically significant associations were observed in genotypic frequencies between cp and apai g / t rs#7975232 ( p=0.939 ) , bsmi a / g rs#1544410 ( p=0.466 ) , and foki a / g rs#2228570 ( p=0.239 ) snps . tables 37 show the numbers of the pcr products ( varied between 93 and 122 samples ) that have been successfully sequenced and read for the analyzed snps . genotypic frequencies of vitamin d receptor gene apai g / t rs#7975232 snp the level of significance ( p<0.05 ) . genotypic frequencies of vitamin d receptor gene apai c / t rs#731236 snp the level of significance ( p<0.05 ) . allelic frequencies of vitamin d receptor gene apai c / t rs#731236 snp the level of significance ( p<0.05 ) . genotypic frequencies of vitamin d receptor gene bsmi a / g rs#1544410 snp the level of significance ( p<0.05 ) . genotypic frequencies of vitamin d receptor gene foki a / g rs#2228570 snp the level of significance ( p<0.05 ) . cp is frequently seen among family members and across different generations within a family , suggesting a genetic basis for the susceptibility to periodontal disease ( 1 ) . in addition , studies of twins suggest a genetic contribution to cp in general , but the effects of bacterial transmission and environmental influences make it difficult to explain a complex interaction ( 1 , 29 ) . for instance , snps of interleukin ( il- ) 1 , il-1 , located in different regions of cytokine genes have also been shown to affect the risk of the disease in several populations ( 30 , 31 ) . because cp is a multifactorial disease ( gene environment interaction ) , several genetic factors may play a role in the disease development by alteration of the structural component of the periodontal tissues , whereas other genetic factors play a role in regulation of inflammatory response ; many gene products interact with each other with environmental factors and the mechanism in which this interaction can increase the risk of cp is incompletely interpreted ( 32 ) . a finding by naito et al . ( 17 ) suggests that the chemical reactions involving bone mineral density - mediated effects are essential for the development of cp , but the effect of the vdr gene polymorphisms in total on cp have not been confirmed ( 2 ) . our results showed a significant association between cp and newly detected apai c / t snp # rs731236 , which is located in intron 8 of the vdr gene . this finding suggests an essential parameter that generally has not been considered on the prevention of diseases related to bone mineral density such as cp . in the present study no significant association was found between cp and apai g / t snp rs#7975232 , bsmi a / g snp rs#1544410 , and foki a / g snp rs#2228570 . our findings agreed with studies on brazilian , turkish , japanese , and chinese populations by de souza et al . ( 6 ) , gunes et al . ( 11 ) , naito et al . ( 34 ) , respectively , whereas other studies showed significant association between these vdr gene polymorphisms and the periodontal disease ( 19 , 35 ) . to our knowledge , the present study is the first study to examine the association between cp and vdr gene polymorphisms in north africa . in this study our results showed a significant association between cp and age , which agrees with a study on by amarasena et al . it is suggested that as the length of time increases , the chronic plaque accumulation increases , followed by increase in periodontal tissues destruction ( 1 ) . the causes of these sex differences have not been explained clearly , but suggest that men have poorer oral hygiene , less positive thinking toward oral health and dental - visit interaction , which agrees with a study by nazish et al . on the other hand , women still have varied periodontal problems due to hormonal disturbance in various decades of life . in addition , not enough studies have been done in developing countries which might have different results as compared to developed countries ( 38 ) . other studies of hormonal effect on periodontal diseases suggested that estrogen hormone in females can affect tooth retention by preventing the alveolar bone resorption ( 3941 ) . 17-estradiol , the primary female sex hormone , may also play a role in promoting periodontal regeneration in an experimental periodontitis model ( 42 ) . therefore , diagnostic periodontal risk assessments like vdr polymorphisms may be useful in the detection of the individuals susceptible for cp . more genetic studies in the future , including analysis of vdr haplotype alleles , are necessary in the prediction of the disease . clearly , studies with enough funding , larger sample size , and in more geographic regions in the country will be beneficial due to the importance of the vdr gene and its relation to the cp and many other human diseases . in the present study , we investigated apai c / t snp rs#731236 that confirmed the importance of the vdr gene in the libyan population . instead of restriction enzyme technique , which was performed in previous studies , we used the sequencing method which is more advanced , more expensive , and capable of revealing more snps in the vdr gene .

abstractthis article is a historiographical exploration of the special forms of knowledge generation and knowledge transmission that occur along local cultural boundaries in the modern neurosciences . following the inauguration of the so - called law on the re - establishment of a professional civil service in nazi germany on april 7 , 1933 , hundreds of jewish and oppositional neurologists , neuropathologists , and psychiatrists were forced out of their academic positions , having to leave their home countries and local knowledge economies and traditions for canada and the united states . a closer analysis of their living and working conditions will create an understanding of some of the elements and factors that determined the international forced migration waves of physicians and clinical neuroscientists in the twentieth century from a historiographical perspective . while i am particularly looking here at new case examples regarding the forced migration during the national socialist period in germany , the analysis follows german - speaking migr neurologists and psychiatrists who found refuge and settled in canada . these individuals form an understudied group of refugee medical professionals , despite the fact that the subsegments of refugee neurologists and clinical psychoanalysts in the united states , for example , have been a fairly well - investigated population , as the works of grob ( 1983 ) , lunbeck ( 1995 ) , or ash and soellner ( 1996 ) have shown . this article is primarily an exploration of the adjustment and acculturation processes of several highly versatile and well - rounded german - speaking physicians , who had received their prior education in neurology , psychiatry , and basic brain research . they were forced out of their academic home institutions and had to leave their clinical research fields as well as their disciplinary self - understanding behind on the other side of the atlantic . backgroundchronic periodontitis ( cp ) is a common oral disease characterized by inflammation in the supporting tissue of the teeth the periodontium , periodontal attachment loss , and alveolar bone loss . the disease has a microbial etiology ; however , recent findings suggest that the genetic factors , such as vitamin d receptor ( vdr ) gene polymorphisms , have also been included.aiminvestigation of the relationship between vdr gene polymorphisms and cp among libyans.materials and methodsin this study , we examined 196 unrelated libyans between the ages of 25 and 65 years , including 99 patients and 97 controls . an oral examination based on ramfjord index was performed at different dental clinics in tripoli and information were collected using a self - reported questionnaire . dna was extracted from buccal swabs ; the vdr apai , bsmi , and foki polymorphisms were genotyped using polymerase chain reaction and were sequenced using sanger method.resultsa significant difference in the newly detected apai snp c / t rs#731236 was found ( p=0.022 ) , whereas no significant differences were found in apai snp g / t rs#7975232 , bsmi snp a / g rs#1544410 , and foki snp a / g rs#2228570 between patients and controls ( p=0.939 , 0.466 , 0.239 ) , respectively.conclusionvdr apai snp c / t rs#731236 may be related to the risk of cp in the libyan population .

acne vulgaris is a very common chronic inflammatory disease of the pilosebaceous glands , which may occur with comedones , papules , pustule forms , cysts , nodules , and scarring in different area of the body , especially face . without proper treatment , it can cause scarring and hyperpigmentation . the prevalence of acne is very different in various countries ; ranging from 0 to over 90% . according to the estimates of the us census bureau international data base ( 2004 ) , among 68 million iranians , approximately its importance should not be ignored because the disease can cause many psychological and social effects for the patient such as low self - esteem , social withdrawal , depression , anxiety , and embarrassment . four main factors involving in the pathogenesis of acne are the production of sebum , severe keratinized ( horny ) of the pilosebaceous duct , disruption of microbial flora , particularly propionate acne bacteria and inflammation . no topical drug has been found to be effective on all these factors , and there is no treatment for complete and permanent improvement of acne . therefore , it seems logical to investigate for simple , uncomplicated and yet effective treatment . chemical peeling was used in different communities for centuries . currently , the most commonly used superficial peelings are glycolic acid , tri - chlorine acetic acid , salicylic acid , pyruvic acid , resorcinol , and solid carbon dioxide . pyruvic acid ( ch3-co - cooh ) is -kato acid with properties such as keratolytic , antimicrobial , the ability to stimulate collagen building , and formation of elastic fibers . pyruvic acid efficacy is known for the treatment of many skin diseases such as acne , superficial scars , photo - damage , and pigment disorders . this study was designed to assess the effect of adding 50% pyruvic acid compared to 30% salicylic acid on the treatment of acne . in a prospective one - blinded clinical trial , 86 patients were selected who referred to al - zahra hospital dermatology clinic and isfahan skin research center within the second 6 months of 2010 ( project no . the inclusion criteria were as follows : ( 1 ) age of 1540 years , ( 2 ) the existence of mild to moderate acne on the face skin ( not more than five pustule forms and no cysts , nodules , and colloidal deep scar observed in the affected area ) , ( 3 ) no history of drug allergy , and ( 4 ) no use of isotretinoin ( at least 6 months before the baseline ) . while the exclusion criteria were as follows : ( 1 ) drug allergy , ( 2 ) recurrent facial herpes simplex , ( 3 ) history of topical or systemic antibiotic therapy , ( 4 ) pregnancy and lactation , and ( 5 ) lack of cooperation or access to the patient . the method , benefits , and side effects of this method have been described for the patients before entering the study , and written informed consents were obtained from them . the patients randomly allocated in one of the two groups under study . in the first group , 43 patients as the control group underwent the routine treatment of acne ( containing erythromycin topical solution 4% made by pak - darou company , triclocarban soap made by golmar company , and fat - free sunscreen with specific pathogen - free thirty made by dr . jila company ) were used twice a day at home . an alcohol - based solution of 30% salicylic acid ( merck , germany ) was applied in the clinic for them every 2 weeks . in the second group , 43 patients as the case group underwent the routine treatment of acne twice a day at home . a 50% hydroalcoholic solution of pyruvic acid ( merck , germany ) was applied for them in the clinic for every 2 weeks . the number of comedones , papules , and pustule forms were counted by a physician for each patient before the treatment ( first visit ) and then at week 2 , 4 , 6 , and 8 ( second to fifth visit ) . the severity of acne on the face of each patient was changed to quantitative data by using the acne severity index ( asi ) and recorded in the data collection list . asi was calculated as follows : asi = ( 0.25 comedones ) + papules + ( 2 pustules ) . the asi was proposed 100% in the first visit regardless of the severity of acne lesions and the percentage decrease in the next visits was considered as the recovery percentage . the followings were considered : improved more than 75% = excellent , improved 5075% = good response , improved 2550% = moderate response , and improved < 25% = poor response . the patients were instructed how to use drugs at home ( avoid direct contact with sunlight , use sunscreen before leaving home , and renewed once every 2 h ) . the procedure was as follows in the case of peeling ( pyruvic acid or salicylic acid ) : at first , the patient 's skin was cleaned ( by impregnated cotton with 70% ethanol)in the next step , the standard solution of pyruvic acid 50% or salicylic acid 30% was applied on the patient 's face with a cotton applicator . so that , the forehead , the sides of the face , cheeks , chin , and nose were impregnatedafter 35 min , the effect of the used solution was returned to neutral with 10% sodium bicarbonate solution in waterthe treatment was continued once every 2 weeks for 8 weeks . at first , the patient 's skin was cleaned ( by impregnated cotton with 70% ethanol ) in the next step , the standard solution of pyruvic acid 50% or salicylic acid 30% was applied on the patient 's face with a cotton applicator . so that , the forehead , the sides of the face , cheeks , chin , and nose were impregnated after 35 min , the effect of the used solution was returned to neutral with 10% sodium bicarbonate solution in water the treatment was continued once every 2 weeks for 8 weeks . patient characteristics , side effects ( itching , erythema , burning , and scaling ) , and patient satisfaction ( excellent , good , fair , poor ) were recorded using a checklist . chi - square test was used to compare the qualitative variables between the two groups . the repeated measurement anova method was used to show the differences between the various stages of the treatment in each group and also between the two groups . in a prospective one - blinded clinical trial , 86 patients were selected who referred to al - zahra hospital dermatology clinic and isfahan skin research center within the second 6 months of 2010 ( project no . the inclusion criteria were as follows : ( 1 ) age of 1540 years , ( 2 ) the existence of mild to moderate acne on the face skin ( not more than five pustule forms and no cysts , nodules , and colloidal deep scar observed in the affected area ) , ( 3 ) no history of drug allergy , and ( 4 ) no use of isotretinoin ( at least 6 months before the baseline ) . while the exclusion criteria were as follows : ( 1 ) drug allergy , ( 2 ) recurrent facial herpes simplex , ( 3 ) history of topical or systemic antibiotic therapy , ( 4 ) pregnancy and lactation , and ( 5 ) lack of cooperation or access to the patient . the method , benefits , and side effects of this method have been described for the patients before entering the study , and written informed consents were obtained from them . the patients randomly allocated in one of the two groups under study . in the first group , 43 patients as the control group underwent the routine treatment of acne ( containing erythromycin topical solution 4% made by pak - darou company , triclocarban soap made by golmar company , and fat - free sunscreen with specific pathogen - free thirty made by dr . an alcohol - based solution of 30% salicylic acid ( merck , germany ) was applied in the clinic for them every 2 weeks . in the second group , 43 patients as the case group underwent the routine treatment of acne twice a day at home . a 50% hydroalcoholic solution of pyruvic acid ( merck , germany ) was applied for them in the clinic for every 2 weeks . the number of comedones , papules , and pustule forms were counted by a physician for each patient before the treatment ( first visit ) and then at week 2 , 4 , 6 , and 8 ( second to fifth visit ) . the severity of acne on the face of each patient was changed to quantitative data by using the acne severity index ( asi ) and recorded in the data collection list . asi was calculated as follows : asi = ( 0.25 comedones ) + papules + ( 2 pustules ) . the asi was proposed 100% in the first visit regardless of the severity of acne lesions and the percentage decrease in the next visits was considered as the recovery percentage . the followings were considered : improved more than 75% = excellent , improved 5075% = good response , improved 2550% = moderate response , and improved < 25% = poor response . the patients were instructed how to use drugs at home ( avoid direct contact with sunlight , use sunscreen before leaving home , and renewed once every 2 h ) . the procedure was as follows in the case of peeling ( pyruvic acid or salicylic acid ) : at first , the patient 's skin was cleaned ( by impregnated cotton with 70% ethanol)in the next step , the standard solution of pyruvic acid 50% or salicylic acid 30% was applied on the patient 's face with a cotton applicator . so that , the forehead , the sides of the face , cheeks , chin , and nose were impregnatedafter 35 min , the effect of the used solution was returned to neutral with 10% sodium bicarbonate solution in waterthe treatment was continued once every 2 weeks for 8 weeks . at first , the patient 's skin was cleaned ( by impregnated cotton with 70% ethanol ) in the next step , the standard solution of pyruvic acid 50% or salicylic acid 30% was applied on the patient 's face with a cotton applicator . so that , the forehead , the sides of the face , cheeks , chin , and nose were impregnated after 35 min , the effect of the used solution was returned to neutral with 10% sodium bicarbonate solution in water the treatment was continued once every 2 weeks for 8 weeks . patient characteristics , side effects ( itching , erythema , burning , and scaling ) , and patient satisfaction ( excellent , good , fair , poor ) were recorded using a checklist . chi - square test was used to compare the qualitative variables between the two groups . the repeated measurement anova method was used to show the differences between the various stages of the treatment in each group and also between the two groups . among 86 patients entered the study , 43 patients ( group 1 ) received pyruvic acid and 43 patients ( group 2 ) received salicylic acid . in group 1 , there were forty women ( 93.02% ) and three men ( 6.98% ) and in group 2 , 39 women ( 90.7% ) and four men ( 9.3% ) were enrolled . the mean age in group 1 was 25.07 6 years and in group 2 , it was 23.05 5.7 years . twenty patients in group 1 ( pyruvic acid ) and 27 patients in group 2 ( salicylic acid completed the 8-week treatment period ) . the reasons for not continuing the treatment by the excluded patients from the study were described in the following categories : family problems ( five patients in group 1 and one patient in group 2 ) , distance to the clinic ( six patients in group 1 and eight patients in group 2 ) , not satisfied with the treatment ( three patients in group 1 and two patients in group 2 ) , and unknown causes ( four patients in group 1 and five patients in group 2 ) [ figure 1 ] . among the people who had completed the study , the average number of the comedones , papules , and pustule forms in each session were calculated separately in the two groups [ table 1 ] . mean and standard deviation of the number of the comedones , papules , and pustule forms in final patients in both groups for each session in both groups , the comparison between the numbers of comedones was observed that during the course of treatment , the number of lesions has been downward , which was statistically significant ( p < 0.001 ) . however , changes in the number of pustule forms did neither increase nor decrease . comparing between the two treatments groups , the effect of two drugs in the change of noninflammatory and inflammatory lesions of acne after reviewing the changes , during the course of treatment , the results showed that the reduction in the number of comedones in the second visit than the first one ( p = 0.002 ) and in the third visit than the second one ( p = 0.019 ) was statistically significant . however , the decrease was not statistically significant in comparison between the fourth and third visits ( p = 0.097 ) and between the fifth and fourth visits ( p = 0.567 ) . reduction the number of papules between the second and the first visits ( p = 0.021 ) , the third and the second visits ( p = 0.019 ) and the fourth and third visits ( p = 0.020 ) was statistically significant . however , this drop compared to the fifth and fourth visits was not statistically significant ( p = 0.137 ) . reduction of the number of pustules was only statistically significant between the second and first visits ( p = 0.010 ) . asi was calculated for each patient at every session , and the percentage changes were obtained over several sessions . comparing these responses in both groups are shown in table 2 . severe lesion was defined as an increase in the percentage of asi . at the end of treatment , the mean percentage of changes in asi was compared in the two groups where the results are given in table 3 . as it can be seen in this table , in both groups between each visit , compared with the previous visits , the average rate of decline in this index was only statistically significant in the second to the first visit ( p < 0.001 ) . moreover , in both groups , the comparison between the values of these averages showed that its decline during the course of treatment was statistically significant ( p < 0.001 ) . by comparing between the two groups , the reduction in the average of asi index had no statistically significant difference and no difference was found in the effect of the two treatments . changes in the recovery rate of patients comparison of changes in the acne severity index between groups the complications due to the peeling with these two treatments were as follows : scaling - regarding the percentage , there were no significant difference between two groups of patients with this complication in the first to fourth sessions . however , in the fifth session , the rate of this complication was significantly lower in the patients treated with salicylic acid ( p = 0.015)redness - there was no significant difference between the two groups during the various sessions in terms of the percentage of the patients with this conditionburning - this complication was occurred in more than 85% of patients in both groups at all of the sessions after using the drug . there was no significant difference between the two groups during the various sessions regarding the number of patients with this conditionitching - in pyruvic acid group , three patients after the first session and one patient after the fourth session faced with this condition . in the salicylic acid group , there was only one patient with this complication in the second session . there was no significant difference between the two groups during the various sessions regarding the number of patients with this complication . scaling - regarding the percentage , there were no significant difference between two groups of patients with this complication in the first to fourth sessions . however , in the fifth session , the rate of this complication was significantly lower in the patients treated with salicylic acid ( p = 0.015 ) redness - there was no significant difference between the two groups during the various sessions in terms of the percentage of the patients with this condition burning - this complication was occurred in more than 85% of patients in both groups at all of the sessions after using the drug . there was no significant difference between the two groups during the various sessions regarding the number of patients with this condition itching - in pyruvic acid group , three patients after the first session and one patient after the fourth session faced with this condition . in the salicylic acid group , there was only one patient with this complication in the second session . there was no significant difference between the two groups during the various sessions regarding the number of patients with this complication . hyperpigmentation occurred in three patients of pyruvic acid group , two of them withdrew the study . in one patient , who continued the treatment , the symptom was resolved after 2 weeks . the patients , who completed the 8-week treatment , finally were asked about their degree of satisfaction . there were no significant difference in patient satisfaction between two treatment groups ( p = 0.62 ) . about 19 patient in pyruvic acid and 17 patients in salicylic acid treated groups reported excellent or good [ table 4 ] . pyruvic acid 50% and salicylic acid peels have several cosmetic indications including skin rejuvenations and the treatment of moderate acne scars . few studies have been performed about the effect of pyruvic acid on acne . in 2002 , a study including forty patients with mild to moderate acne was conducted by cotellessa et al . in italy . after applying pyruvic acid 4050% , 16 patients ( 40% ) had complete remission , twenty patients ( 50% ) had partial improvement , and in four patients ( 10% ) had no recovery was observed . the transient erythema and mild scaling was evident in all patients . in 2004 , another study with forty patients was performed by pacifico et al . in italy with similar results and no side effects were observed in their study as well . in 2006 , a study by tosson et al . took place in egypt . in response to pyruvic acid 4050% , among thirty patients with acne , 33.3% had very good response ( complete healing ) , 20% had a good response ( more than 75% healing ) , and 36.7% had a moderate response ( 5075% improvement ) . in their study , a clear relation was observed between acne severity and the response to pyruvic acid . the patients with mild acne had revealed much better response to the treatment than the other patients . mild scaling after each peeling session with pyruvic acid lasted for 710 days , and it did not interfere with social activities of the patients . another study has been conducted by berardesca et al . on twenty patients with photo - damage , superficial scars , and melasma in 2006 . after applying pyruvic acid 50% , significant reduction was observed in the degree of skin pigmentation in patients with melasma . in addition to this , there was a significant increase in skin elasticity in all patients . no cases of hyperpigmentation or erythema after the inflammation ( proximal interphalangeal ) were observed . in iran , no study has been performed on the effect of pyruvic acid on acne . in previous studies mentioned above the effects of pyruvic acid 50% were compared with salicylic acid 30% . on the other hand , there were vague hints in studies conducted in other countries . for example , the criteria to classify acne in mild , moderate and severe were not clearly described in the studies conducted in italy . in a study took place in egypt , the canadian association of general surgeons has been used as the measuring scale for determining the severity of acne . in this scoring , the value of all different skin lesions is the same and the number of the lesions are not important . there were statistically significant reductions in number of comedones and papules during the treatment in pyruvic acid - treated patients in our study in contrast to the number of pustules . findings regarding an inverse relationship between acne severity and response to pyruvic acid . in our study , in both groups , the more the peeling sessions , the less the reduction in number of lesions was . significant reduction in facial sebum secretion is reported following both 50% pyruvic acid and 30% salicylic acid peels , which could be a contributing mechanism in the efficacy of these agents in acne improvement . the most common side effects of pyruvic acid and salicylic acid peeling were burning , redness , and scaling . in each session , the burning occurred in 85100% of the patients which was resolved immediately by using sodium bicarbonate 10% as it was mentioned in tosson et al . 's study . unlike the study conducted by cotellessa et al . , the scaling and redness did not occur in all patients treated with pyruvic acid . it can be concluded from this study that the efficacy of adding pyruvic acid 50% and salicylic acid 30% to common treatment of mild to moderate acne is similar in terms of reducing the number of inflammatory and noninflammatory acne lesions . side effects of both peelings were also similar , except for the scaling in the fifth session that was significantly lower in the patients treated with salicylic acid frequent peeling sessions that resulted in lower cooperation of the patients and withdrawal from the study was the most important limitation . further clinical trials with less peeling sessions and more follow - up periods are recommended to evaluate the best effective interval of subsequent peelings and duration of acne improvement after the therapeutic plan . fj contributed in the conception of the work , analysis and interpretation of data for the work , drafting and critical revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work.gf contributed in the conception and design of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work.ss contributed in the conception of the work , acquisition of data , drafting the work , approval of the final version of the manuscript , and agreed for all aspects of the work.smh contributed in the design of the work , analysis of data , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . fj contributed in the conception of the work , analysis and interpretation of data for the work , drafting and critical revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . gf contributed in the conception and design of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . ss contributed in the conception of the work , acquisition of data , drafting the work , approval of the final version of the manuscript , and agreed for all aspects of the work . smh contributed in the design of the work , analysis of data , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . hermansky and pudlak reported in 1959 two cases of unrelated albinos with lifelong bleeding tendency and peculiar pigmented reticular cells in the bone marrow as well as in lymph nodes and liver biopsies . one albino was female and the other was male , both were 33 years old . after her death , autopsy revealed horseshoe kidney with large amount of pigment in reticuloendothelial cells and in the walls of small blood vessels . hermansky - pudlak syndrome ( hps ) is an autosomal recessive disorder that is caused by different genetic mutations . this syndrome is characterized by oculocutaneous albinism , platelets storage - pool deficiency , and lysosomal accumulation of ceroid lipofuscin . these patients have decreased skin and hair pigmentation and transillumination of the iris with markedly decreased visual acuity . patients with hps have easy bruisability , secondary to the absence of platelet dense bodies , which trigger platelet aggregation . ceroid lipofuscin is an amorphous lipid protein complex , and its accumulation in tissues is believed to be responsible for pulmonary fibrosis and granulomatous colitis . this ceroid lipofuscin also accumulates in the kidney , giving it a dark appearance when examined during an autopsy or kidney biopsy [ 4 , 5 ] . although no specific renal disease has been attributed to hps , compromised renal function in these patients has been reported [ 1 , 4 ] . renal insufficiency has been thought to occur due to the ceroid lipofuscin deposition in the kidney . gahl et al . reported a series of 49 patients with hsp , of which 9 had ckd ; however , no kidney biopsies where performed and there is no information about the proteinuria and hypertension in any of the above reports . to our knowledge , we report the first pediatric renal pathology case of hps associated with ckd , tubular pathology , focal segmental glomerulosclerosis ( fsgs ) , and hypertension . the patient is a 15-year - old male born in puerto rico to nonconsanguineous parents of puerto rican descent . he moved to the usa when he was 2 years old at which time the diagnosis of hps was made by hematologist who saw him because of easy bruisability and prolonged bleeding time . genetic testing was done , and he was found to be homozygous for the 16 bp duplication in the hps1 gene , which is denoted c.1472_87dup 16 on the cdna level . he had no known family history in parents , grandparents , and immediate relatives of renal failure , albinism , or known hps , although paternal grandmother had hypertension . our service was consulted secondary to persistent hypertension detected during a hospitalization for asthma - like symptoms , when he was 8 years old . physical examination at that time showed an active child , with global developmental delay , albino , obese ( with body mass index > 95th for age ) with normal cardiovascular and respiratory exam . he had no fever , heart rate was 96 beats per minute , and blood pressure in the right arm was 149/93 mmhg , left arm 127/78 mmhg , right leg 154/64 mmhg , and left leg 151/77 mmhg in sitting position . serum chemistries showed sodium of 135 mg / dl , potassium of 3.8 mg / dl , blood urea nitrogen of 16 mg / dl , and creatinine of 1 mg / dl . renal ultrasound showed both kidneys to be normal in position and without evidence of hydronephrosis . the right kidney measured 8.4 cm , and left kidney measured 9.7 cm . doppler examination demonstrated patency of renal veins , with resistance indices 0.5 ( right renal artery ) and 0.6 ( left renal artery ) . his hypertension workup included normal serum / urine catecholamines , thyroid function tests , and normal brain imaging ( mri head ) . to control hypertension , , patient 's blood pressure was < 90 percentile for age , gender , and height . lvh subsequently resolved as documented on follow - up echo at age of 9 years . at 12 years of age , calcium channel blocker was stopped , secondary to gingival hyperplasia , and an angiotensin receptor blocker ( arb ) was started . at that time , patient did not have proteinuria . his serum creatinine remained stable ( ranged from 1 to 1.3 mg / dl ) , and serum potassium ranged from 3.8 to 4.6 mg / dl . in this time period , , he had a gastric , duodenum , and colonic biopsies done , which showed mild acute - chronic inflammation ( there were no bleeding complications ) . during his routine follow - up visit at age 14 years , 30 mg / dl of protein without hematuria was noted on the dipstix , but measured urine protein / creatinine ratio ( upc ) of this sample was 0.2 . he was seen in the renal clinic every 36 months , and since then his first morning upc ranged from 0.2 to 0.38 . a reliable 24-hour urine collection was not possible to obtain due to his developmental delay . when he was 15 years old , his blood pressure was still controlled with arb , his serum creatinine was elevated at 1.4 mg / dl , and proteinuria became fixed , that is , 1st morning upc was 0.38 and 0.5 . first morning urinalysis showed ph of 6 , yellow color , specific gravity 1.020 , 100 mg / dl protein , no blood , and no glucose , and microscopic examination showed 03 red blood cells per high - power field and 05 white blood cells per high - power field , without casts , indicative of functional renal concentrating and acidifying abilities . he had no edema and no hematuria , and his serum albumin was 4.2 g / dl . his serologies for hepatitis b / c and lupus ; complement 3 and 4 , antinuclear antibodies , and vasculitis ; antineutrophil cytoplasmic antibodies ( anca ) were all negative . kidney biopsy was performed due to persistent proteinuria and compromised renal function , with estimated gfr 70 ml / min/1.73 m by schwartz formula . the hematology service was consulted prior to biopsy and coagulation studies : prothrombin time ( pt ) was 10.5 seconds ( normal 9.512 seconds ) and partial thromboplastin time was 26.9 seconds ( normal 26.233.8 seconds ) . immediately after the kidney biopsy , a small perinephric hematoma was observed by ultrasonographic evaluation but no gross hematuria was seen and there were no other complications during and after the procedure . tissue for light microscopy was evaluated at 10 section levels with h&e , pas , and trichrome staining . there were total 19 glomeruli in two renal biopsy cores of tan - red cylindrical tissue each core up to 1.2 cm and 1.9 cm . there were three sclerotic glomeruli , including one with segmental hyalinosis ; one of the four additional enlarged glomeruli per level of section had perihilar segmental sclerosis . mild interstitial fibrosis with associated tubular atrophy , and no significant interstitial inflammation was present . acute tubular injury with variable tubular ectasia , variable hypertrophic tubular epithelial cells with marked cytoplasmic vacuolization , fuchsinophilic and pas - negative waxy dull brown - yellow spherule cytoplasmic inclusions were present . no arteriosclerosis was present , and no vascular inflammatory lesions were present , figures 1(a)1(c ) . on immunofluorescent examination , up to 3 enlarged glomeruli per level of section had irregular coarse granular capillary wall and mesangial staining with antisera specific for c3 . the glomeruli had no staining with antisera specific for igg , iga , igm , c1q , kappa light chains , or lambda light chains . electron microscopy ( figure 2 ) revealed no capillary wall or mesangial immune complex - type electron - dense deposits . although original description of hps originated in prague , the majority of literature reports describing this syndrome have come from puerto rico . when hermansky and pudlak reported for the first time in 1959 two cases of albinism and prolonged bleeding time , renal insufficiency was described in one patient with horseshoe kidney . tagboto et al . reported a patient with hps with iga nephropathy and anca - positive glomerulonephritis . the kidney tissue showed a dark - brown pigmentation , and the tubular injury was similar to what we found in our case . ceroid - like lipofuscin material has been found in the urine of hps patients with renal involvement , and the deposition of this material in other tissues ( lungs , colon ) has been blamed for cellular dysfunction and subsequently fibrosis , although , if this is the cause , the exact mechanism has not been described . the mechanism of injury of renal tubular cells by lipofuscin accumulation is not completely understood . hps is a rare , autosomal recessive disorder which affects multiple cytoplasmic organelles : melanosomes , platelet dense granules , and lysosomes . hps can be caused by mutation in several genes : hps1 ( common among population of the northwestern part of puerto rico ) , hps3 , hps4 , hps5 , and hps6 . hps2 , which includes immunodeficiency in its phenotype , is caused by a mutation in the ap3b1 gene . hps is characterized by defect in the coat - protein complexes resulting in abnormal function of lysosomes , melanosomes , and platelet dense granules as hps - gene products are part of distinct protein complexes : the adaptor complex ap-3 and various blocs ( biogenesis of lysosome - related organelles complex ) [ 1416 ] . although there is no data to support it , it is plausible to hypothesize that podocytes of hps subjects may demonstrate molecular defects in proteins analogous to dysbindin ( part of bloc-1 , found in axonal synapses ) involved in lysosomal trafficking . the hps phenotype is heterogenic , and it was reported that hps observed in an isolated swiss alps village [ 17 , 18 ] usually shows a relatively mild clinical course with normal life expectancy and the lack of clinical manifestations of ceroid storage . our patient presents yet another phenotype with htn and constellation of renal lesions that include evidence of tubular injury and fsgs . patients with hps have mild prolonged bleeding time secondary to functionally impaired platelets , but platelet numbers are normal . our patient had had a gastric and colonic biopsy in the past , as well as a tooth removal without significant bleeding . slow rise in serum creatinine from 1 mg / dl at age 8 y to 1.4 mg / dl at age 16 y might be clinically important in our case description , indicative of slow progression despite multiple risk factors : obesity , htn , fsgs , and presence of tubular fuchsinophilic cytoplasmic inclusions with mild interstitial fibrosis on renal biopsy . to the best of our knowledge , there are no previous reports of pediatric hps patients with described renal pathology . the animal model of hps is the fawn - hooded rat ( fh ) and pale ear mouse [ 19 , 20 ] . the fh rat has an inherited platelet storage - pool deficiency and a widespread impairment of serotonin storage , susceptibility to systemic and pulmonary hypertension , but does not have mutations in the gene homologous to hps1 . the fh rat has a genetic predisposition to develop chronic renal failure , fsgs , htn , and proteinuria . the progression of sclerosis results in premature death from end - stage kidney disease in these rats . but the precise cause of the spontaneous development of systemic hypertension in fh rats is not well understood . hps1 is homologous with the pale ear mouse ( ep mutation ) . in that mouse , there is lysosomal accumulation of beta - galactosidase and em showed large multilamellar granules in proximal tubules with the accumulation of lipofuscin material in the tubules . in the last decade , verani reported 14 biopsies of obese adults with fsgs . in that report , glomerulomegaly was reported in patients with fsgs associated with obesity , as well as lack or hyperplasia of glomerular epithelial cells . in that report , patients with fsgs associated with obesity had higher cholesterol and atherosclerosis . our patient had normal cholesterol and no atherosclerosis on biopsy . no tubular lesions , as in the case of our patient , had been reported in fsgs associated with obesity . in a previous case of hps , tubular atrophy and accumulation of a ceroid - like material in tubular epithelial cells were reported . our patient had increased bmi and hypertension with lvh since initial presentation at age 8 years . we can speculate that htn was secondary to obesity at 8 years of age but it would be uncommon clinical scenario to encounter the child at this age with htn significant enough to cause lvh due to obesity alone . our patient did not have evidence of renal artery stenosis or endocrine causes of hypertension , and his blood pressure was controlled with monotherapy . the effects on the kidney secondary to obesity in animals and humans include structural and functional changes , such as increased gfr , increased renal blood flow , and renal hypertrophy [ 21 , 22 ] . some of the changes in the renal tissue of our patient might be consistent with fsgs associated with obesity , and the tubular changes may contribute to the renal insufficiency . although it is not our intention to draw any association between hps and fsgs , we can speculate that obese patients with hps and hypertension may be at risk of unrecognized fsgs . however , it is possible that the sclerosis of the glomeruli observed in our case is secondary to the tubular damage . to the best of our knowledge , this is the first pediatric case of hps associated with ckd and defined renal pathology . further studies are needed to determine the cause of renal insufficiency in patients with hps .

background : acne vulgaris is a chronic inflammatory disease of the pilosebaceous follicles and one of the most common skin diseases . the peeling method has been recently found to be effective for acne treatment . this study aimed to compare the efficacy of pyruvic acid 50% and salicylic acid 30% peeling in the treatment of mild to moderate acne.materials and methods : in a prospective single - blinded clinical trial , 86 patients with acne were randomly assigned into two groups . in both groups , the routine treatment of acne ( topical solution of erythromycin 4% , triclorocarban soap , and sunscreen ) were used twice a day for 8 weeks . in addition , salicylic acid 30% for the control group and pyruvic acid 50% for the case group were used . in both groups , acne severity index ( asi ) was calculated before and at week 2 , 4 , 6 , and 8 of the treatment . patient satisfaction was assessed at the end of the treatment . side effects were recorded using a checklist.results:in both groups , the reduction in the number of comedones , papules , and asi were statistically significant ( p < 0.001 ) in the course of treatment . however , it was not significant regarding the number of pustules ( p = 0.09 ) . none of the number of comedone , papules , pustules , and asi was statistically different between study groups . both treatment groups had similar side effects except for scaling in the fifth session , which was significantly lower in salicylic acid treated patients ( p = 0.015).conclusion : both pyruvic acid 50% and salicylic acid 30% are effective in the improvement of mild to moderate acne with no significant difference in efficacy and side effects . we report a child with hermansky - pudlak syndrome ( hps ) and chronic kidney disease ( stage ii ) with histological diagnosis of focal segmental glomerulosclerosis ( fsgs ) . a 15-year - old male of puerto rico ancestry with history of hps , hypertension ( htn ) , asthma , obesity , and chronic kidney disease ( ckd ) stage ii presented with new - onset proteinuria without edema . his blood pressure had been controlled , serum creatinine had been 0.91.4 mg / dl , and first morning urine protein / creatinine ratio ( upc ) ranged from 0.2 to 0.38 . due to persistent nonorthostatic proteinuria with ckd , renal biopsy was performed and fsgs ( not otherwise specified ) with chronic diffuse tubulopathy ( tubular cytoplasmic droplets ) and acute tubular injury was reported . ceroid - like material is known to infiltrate tissues ( i.e. , lungs , colon , and kidney ) in hps , but the reason for the renal insufficiency is unknown . nonspecific kidney disease and in one adult case iga nephropathy with anca - positive glomerulonephritis have previously been reported in patients with hermansky - pudlak syndrome . to our knowledge , we report the first pediatric renal pathology case of hps associated with ckd . this paper discusses presentation and management of renal disease in hps .

a 60-year - old gentleman presented to our institution with pain and dimness of vision in both eyes . on evaluation , his best corrected visual acuity ( bcva ) was 20/80 in right eye ( re ) and 20/120 in left eye ( le ) . his cup - to - disc ratio was 0.6 re and 0.85 le with loss of the neural rim . he was prescribed betoxolol 0.5% in both eyes ( be ) and recommended elective cataract extraction for the le . keratometry measurements were 43.75 diopter ( d ) 180 and 40.00 90 os with 3.75 d of against the rule astigmatism . the calculated intraocular lens ( iol ) power was 23.50 d. the patient had used topical ofloxacin 3 mg / ml six times , 1 day prior to surgery , and thrice on the day of surgery at 1-hour intervals . povidone iodine 5% was used half an hour before surgery and just before the commencement of surgery . ocular adnexa was cleaned using 10% povidone iodine , the patient was draped and speculum was applied . a manual sutureless cataract extraction ( mscs ) with lri was planned for the le . through a 6-mm temporal sclerocorneal tunnel , a nasal 6-mm lri was performed using a 550-m steel knife prior to wound construction . on the first postoperative day , his bcva le was 20/60 , and the anterior segment was unremarkable except for mild iritis . on the 15 postoperative day , he presented emergently with pain and redness in the operated eye . on examination , anterior segment examination revealed circumcorneal congestion , iris nodule , a 1.5-mm hypopyon , and a corneal exudate at the site of the prior lri [ fig . 1 ] . b - scan ultrasonography revealed minimal vitreous exudates . on presentation at 15 post - op day with endothelial nodule at the site of limbal relaxing incision , hypopyon and iris nodule vitreous was aspirated and sent for analysis . intravitreal vancomycin 1 mg / ml and amikacin 400 g/0.1ml were administered . based on the clinical picture of corneal exudate , iris nodules , and hypopyon with minimal posterior segment involvement , a clinical diagnosis of nocardial endophthalmitis was made . the patient was treated with topical gatifloxacin and amikacin hourly , prednisolone acetate every 6 hours , and oral gatifloxacin . the isolate was sensitive to amikacin , gatifloxacin , moxifloxacin , ciprofloxacin , ofloxacin , and chloramphenicol . the inflammation worsened , exudates from the iris extended into the capsular bag and fresh exudates appeared at the sclerocorneal wound site . a subsequent ultrasound revealed increased vitreous opacities . on the 18th postoperative day , the patient underwent core vitrectomy with anterior chamber washout and capsular bag removal with iol explantation . his symptoms improved , iris and endothelial nodules resolved , hypopyon decreased and exudate at the lri site resolved [ fig . 2 ] . he was discharged after a 1-week course on a regimen of oral gatifloxacin and topical amikacin , ciprofloxacin , and a cycloplegic . four weeks later , the patient 's vision was 20/2000 and his examination revealed 2 + white blood cells ( wbcs ) in the anterior chamber , no hypopyon , and vitreous opacities . lri preserves the perfect optical qualities of the cornea and is an excellent option for low - to - moderate degrees of astigmatism in a planned single bioptic procedure . lri produces lesser effect than corneal relaxing incision ( cri ) , thus precise alignment of the axis is not as critical . have shown that lri performed during phacoemulsification surgery is a safe , effective , and stable procedure to reduce pre - existing corneal astigmatism . we are unaware of any prior report of a postoperative endophthalmitis associated with an lri performed at the time of cataract surgery . in this patient , as the lri was of partial thickness and cornea was not perforated , it appears that bacteria may be able to penetrate the cornea at the site of the lri . this patient presented with iris nodules and hypopyon , a classic presentation of nocardial endophthalmitis . the endothelial exudates were at the lri site while the wound was clear , thereby indicating the lri as the probable site of entry for the organism . the rate of postoperative endophthalmitis after cataract surgery in this population is 0.064% and norcardial infections account for 16.4% of these cases . norcardia endophthalmitis is more evident in emaciated and immunocompromised individuals and associated with poor prognosis . mscs is practiced widely in india and constitutes about 62% of the total 69,479 cataract procedures done at our institute in 2008 . the endophthalmitis rate with the mscs group was 0.03% and was lesser than the 0.05% infection rate with the phacoemulsification group ( unpublished data ) . have proved that poorly constructed wounds with a short corneal valve have a higher risk of developing endophthalmitis . in this patient , the scleral and corneal valves were 2 mm and 1.5 mm , respectively . in spite of making a larger incision in the mscs group , the infection rates are probably comparable due to the wider sclerocorneal tunnel covered well with conjunctiva at the end of surgery . lalitha et al . from the same population reported nil endophthalmitis in the 4275 cases who underwent mscs technique in the total group of 36,072 cataract procedures where the overall endophthalmitis rate was 0.05% . in our experience , the mscs technique per se has not been found to be a significant risk factor for endophthalmitis compared to phacoemulsification . in summary patients and clinicians can use it to plan for end of life decisions , such as setting treatment priorities . all of these uses presuppose that an accurate measure of prognosis exists . in this paper , we describe and report on the accuracy of the multimorbidity ( mm ) index . alemi and colleagues16 developed the mm index to account for the prognosis of patients with multiple diagnoses . charlson and colleagues were among the first group of investigators to do so.7 these authors developed an index that predicted mortality from 22 broad disease categories , including one category for all heart diseases , another for aids , and still another for all cancers . deyo et al.,8 romano et al.,9 manitoba et al.,10 and dhoores et al.11 attempted to improve on the initial charlson index by modifying the broad categories and dropping or adding new categories . elixhauser and colleagues12 continued these modifications by creating a list of 30 broad categories of comorbidities , and van walraven et al . organized these categories into an index.13 in contrast to existing approaches , the mm index does not classify diagnoses into broad disease categories . instead , it scores the underlying disease . in existing methods , diagnoses with widely varying hazard ratios secondary malignancies category that are used in variants of the elixhauser index . in a recent study of the prognosis of heart failure patients,14 patients who also had a secondary malignant neoplasm of brain and spinal cord had an odds ratio of mortality equal to 17.28 . in comparison , those who had another variant of a secondary malignancy ( i.e. , a secondary neuroendocrine tumor of distant lymph nodes ) only had an odds ratio of mortality equal to 2.43 . this example illustrates that the same category , secondary malignancies , includes diagnoses that have a nearly ninefold difference in mortality , and that grouping all secondary malignancies into one category overly simplifies the differences within this category . one would anticipate that the accuracy of prognostication could be improved by considering the 28 types of secondary malignancies separately rather than together as one category , which is a feature of the mm index . in this paper , we review the design of the mm index , compare its performance to other prognostic indices , and describe how the index can be used within an electronic health record ( ehr ) . the paper also includes code for constructing the mm index in different data sets or for incorporating the method into ehrs . the key feature of the mm index is that it is built from thousands of diagnoses , without classifying these diagnoses into categories . in order to effectively model the relationship between thousands of diagnoses and mortality , one assumes that the impact of each disease on mortality is independent from other diseases . this assumption is also made in traditional statistical approaches that use linear logistic regression . even though the assumption is obviously false , numerous studies have shown that the naive bayes produces predictions that are as accurate as more complicated models that assume interactions among diseases.1526 in this approach , the overall probability of mortality is calculated as follows : patient 's mm score patient'sdiagnosesldiagnosiswhere ldiagnosis indicates the likelihood ratio associated with the diagnostic code in the training data set . the likelihood associated with each diagnosis is calculated using the following formula from the portion of the data set aside for training of the model : ldiagnosis = prevalenc of diagnosis among patientsprevalenc of diagnosis among alive patients = p(dx|dead)p(dx|alive ) . the likelihood ratio associated with each diagnosis can be interpreted as the number of times the diagnosis increases the odds of mortality . a likelihood ratio of 2 indicates that the diagnosis doubles the risk of mortality , while a likelihood ratio of 0.5 indicates that the odds of mortality is reduced by half . these likelihood ratios were estimated from data within the united states department of veterans affairs ( veterans affairs ) medical records and are publicly available for heart failure1 and for nursing home patients.2 to facilitate the estimation of these ratios for other populations , the standard query language ( sql ) code that we had used is provided in the appendix a. one problem with the method used to estimate the likelihood ratios is that it is affected by co - occurring diagnoses . thus , hypertension may receive higher likelihood of mortality if it tends to occur with more serious diseases such as heart failure or shock . procedures to remove confounding are available in several published papers.27 we recommend the use of stratified covariate balancing ( see r package stratifiedbalancing ) in removing confounding as this approach is not parametric and can be done within ehrs using sql.31 applying methods of removing confounding to the estimation of survival of patients with co - occurring diagnoses remains an active area of research . to derive the mm index , it is important to recognize that a large number of parameters are estimated . there are in excess of 14,000 international classification of diseases ( icd ) codes , and within most populations 3,0005,000 unique diagnoses occur . there are a number of ways to estimate the sample size that would be needed for such a large number of determinations . some investigators have suggested that the power of the investigation depends on the ratio of the number of subjects to the number of variables , using heuristics such as 10 times32,33,34 or 20 times35 the number of subjects compared to the number of variables in the model ( i.e. , in order to estimate 5,000 parameters , 100,000 subjects would be needed ) . in most of the analyses reported here , the total sample size exceeds 30 times the number of variables , suggesting the estimated model has sufficient power to detect the needed parameters . others posit that the most important consideration is the accuracy of local estimates , such as the likelihood ratio associated with each diagnosis.36,37 to calculate those likelihood ratios , a sample size formula for a two - sample t - test can be applied to calculate the required sample size for one diagnosis . in multivariate analysis , the impact of a single diagnosis is evaluated within the context of many others . in these situations , a correction factor associated with the percent of variation explained by the covariates is added to inflate the variance and improve the power.38,39 in selecting one of these two approaches , one may consider the intended use of the study findings . if an overall index score is of interest , then heuristics that use the ratio of the number of subjects to the number of estimates may be most appropriate . however , if individual likelihood ratios associated with each of the respective diagnoses are of concern , then a more localized estimation of the individual likelihood ratios is needed . many investigators and clinicians may wish to estimate the probability of mortality at different time intervals ( e.g. , 6 months , 12 months , or 5 years ) . by using prior odds of mortality , one can transform the mm index to estimate the probability of mortality within a specific period . according to the bayes formula , the posterior odds of mortality are calculated as follows : posterior odds = prior odds*mm indexprobability of mortality = posterior odds1+posterior odds many common diseases are associated with no patient mortality , and there are also rare diseases where every patient dies . in both of these situations , alemi40,41 proposed using the following formulas : ldiagnosis={if all survive1/(n+1)if none surviven+1where n indicates the number of patients with the diagnosis . other adjustments have been reported in the literature , including adding a fraction of a case to either the denominator or the numerator to avoid division by zero . the adjustment used here has the advantage that it is proportional to the number of patients with the diagnosis ( i.e. , a higher likelihood ratio is assigned when all patients die from diseases that are more frequent ) . for example , if all 100 patients with a disease died , then the assigned likelihood ratio is 101 . this assigned likelihood ratio ( i.e. , 11 ) is higher than if all 10 patients with a disease had died . although the mm index is derived from a large data repository , there are several diagnoses that are rare and have insufficient observations to estimate a likelihood ratio . in a minority of cases ( e.g. , when a patient presented with a diagnosis that was not seen in at least 29 cases in the training set ) , then the likelihood ratio associated with a broader diagnostic category is used to score the patient . a typical international classification of disease , revision 9 ( icd-9 ) diagnosis is represented as a five - digit number consisting of three initial digits , a period , and two additional digits . the first three digits represent a disease category . each additional digit after the period represents further refinements . if the patient s diagnosis is rare , then one could use the likelihood ratio for a broader category of the diagnosis that repeats more often ( e.g. , by dropping the last digit in the diagnosis code ) . appendix a includes the sql code that can be used to estimate the likelihood ratios associated with three- , four- , and five - digit codes from data in ehrs . to date , the mm index has been evaluated using diagnoses coded with icd-9 . in the international classification of diseases , revision 10 ( icd-10 ) , a sixth digit was added to further clarify the disease categories . the procedures described in this paper and the computer code provided in appendix a can be used to estimate the prognosis of each code within icd-10 . because icd-10 has tenfold more codes than icd-9 , reliable estimates for this version can not be made until tenfold larger data are available . even when the data are available , many disease codes in icd-10 are unlikely to occur with sufficient frequency so that the prognosis for these codes can be estimated reliably . when icd-10 codes can not be estimated reliably , investigators should combine data and rely on higher order codes in icd-9 , using the procedures explained earlier for estimating rare diseases . if icd-10 codes can be estimated reliably , then these codes should be used instead of icd-9 . by using this method , the best description of the patient would be used . when the estimate is not available , a less precise description would be used . because mm index scores each diagnosis , this leads to a model with thousands of independent variables . since clinicians have a difficult time tracking thousands of variables , a solution is needed that would allow the scoring of each diagnosis but would classify the respective diagnoses into a smaller set of dimensions that are easier to manage . similarly , when evaluating the comparative effectiveness of treatments , there is a need to have sub - indices that indicate the relative severity of different diseases within body systems . the propensity scoring then adjusts for risks introduced by the different body systems and not just the overall mortality risk . the multidimensional multiple morbidity ( mmm ) index was created to address this scenario by breaking the overall mm index into distinct subcategories . the first application of utility theory to prognosis modeling can be traced to the work of gustafson and colleagues.42 later applications have included testing of preferential independence assumptions of additive or multiplicative utility models.43 in this approach , each attribute describes a set of comorbidities , typically within the same body system . the overall mmm score is then organized using the following formulas : mmm=1-i(1=kiui)where : ui is the maximum relative severity of disease in the i category of disease . these scores are scaled to range from 0 to 1 , with 1 indicating the score for the worst disease in the category . these scores are made proportional to the hazard rate associated with each disease and are estimated from the data.ki is the largest probability of mortality associated with the unconfounded impact of diseases within the i category . these scores are scaled to range from 0 to 1 , with 1 indicating the score for the worst disease in the category . these scores are made proportional to the hazard rate associated with each disease and are estimated from the data . ki is the largest probability of mortality associated with the unconfounded impact of diseases within the i category . table 1 shows examples of how the mmm index is organized from a progression in three body systems . three body systems are shown in table 1 , and more are available through the first author.44 the columns in the table show the various body systems ( e.g. , diseases in the circulatory system ) . each row in table 1 corresponds to a particular level of severity . 1st [ hospitalization for ] 459.2 , ruptured abdominal aortic aneurysm . it is assigned a score of 0.4 , while 2 [ hospitalization for ] 785.51 , cardiogenic shock is assigned a score of 1 . in essence , the utility scores list the diagnoses within the system ( from relatively benign to the most serious ) in the order of their hazard rate . this parameter k is the maximum unconfounded probability of mortality within each body system . in this scoring of the body system , each disease is initially rated based on its associated utility score . then within each body system , the health care literature is replete with studies that report the use of physiological markers as prognostic indicators . in this section , alemi and colleagues examined the prognosis of patients with hiv / aids by using an mm index45 in 1999 and found that the index was more predictive of patients survival than an index developed from an average of physiological indicators , such as cd4 t lymphocytes counts . subsequent analyses by other investigators also demonstrated that the index was accurate in predicting hiv / aids prognosis for patients who died in six months and who were eligible for hospice care.46 this early attempt at constructing an mm index did not take into account all diagnoses and was limited to specific diagnoses that followed a diagnosis of hiv / aids . in 2013 , a comprehensive mm index was used to predict 12-month mortality for diabetes patients.47 this index correctly predicted the mortality of 89.9 percent of cases . in as yet unpublished data,48 the performance of the diabetes mm index was compared to the accuracy of hemoglobin a1c levels for 468,867 diabetic patients . a number of investigators have shown how tight control of , or high levels of , hemoglobin a1c might affect prognosis.49,50,51,52,53 figure 1 shows that the sensitivity and specificity of the mm index was superior to prognostic indicators based on hba1c levels alone . the area under an roc curve for predicting 6-month mortality for hba1c levels was 0.652 ; in contrast the roc for the mm index was 0.812 . the mm index was therefore 1.25 times more accurate than the widely used hba1c levels . in 2015 , the mm index was used to predict the 6-month mortality of 140,699 nursing home residents.54 the cross - validated accuracy of the mm index was compared to a common measure of daily functional activity , the barthel index.55,56 the cross - validated roc for the mm index was 0.838 . also in 2015 , the mm index was compared to the ejection fraction for 602,050 unique heart failure patients across 130 veterans affairs medical centers.57 the cross - validated roc for the mm index was 0.784 . in contrast , the cross - validated roc for the ejection fraction was only 0.533 , which was barely above a random chance event . ejection fraction was not predictive of long - term mortality rates . in a study of patients in intensive care , we compared the accuracy of the mm index to 13 physiological markers.58 these markers included the following : ( 1 ) sodium , ( 2 ) blood urea nitrogen , ( 3 ) creatinine , ( 4 ) glucose , ( 5 ) albumin , ( 6 ) bilirubin , ( 7 ) white blood cell count , ( 8) hematocrit , ( 9 ) pao2 , ( 10 ) paco2 , ( 11 ) ph , ( 12 ) egfr , and ( 13 ) lactic acid . we examined the 6-month and 12-month mortality of 442,692 unique patients seen in 87 medical intensive care units of veterans affairs medical centers between 2003 and 2013 . the mm index , relying solely on diagnostic codes , yielded an roc of 0.84 . in contrast , the logistic regression based on the combined impact of 13 physiological markers yielded a roc of 0.65 . these studies collectively show that the mm index is more accurate in predicting 6-month or 12-month mortality compared to select physiological makers . this section compares the mm index and its variants to the charlson index and other diagnoses - based indices . in 2002 , the mmm index was used to predict mortality from childhood diseases.59 the source of data was the 2006 kid s inpatient database of the healthcare cost and utilization project of the agency for healthcare research and quality . this database contained data on pediatric ( children 20 years of age and younger ) discharges from 3,739 community , nonrehabilitation hospitals in 38 states . the accuracy of the mmm index was compared to all patient refined diagnosis - related groups,60 the all payer severity - adjusted diagnosis - related groups,61 and a simple count of diagnoses.62 the mmm index explained 32 percent of the variation in mortality , as measured by percent of deviance explained in mortality . it was fivefold more accurate than all patient refined diagnosis - related groups and threefold more accurate than all payer severity - adjusted diagnosis - related groups . this study demonstrated that prognostic information can be significantly improved by scoring each diagnosis separately . in 2015 , the mm index was directly compared to the charlson and elixhauser variants by predicting the 6-month mortality of heart failure patients.63 in this study , the prognosis of 602,050 unique heart failure patients across 130 veterans affairs medical centers was studied . the mm index was more accurate than the quan variant of the charlson index,64 with an roc of 0.656 . it was also more accurate than the enhanced65 deyo variant66 of the charlson index , with an roc of 0.677 . it was more accurate than the von walraven variant of the elixhauser index,67 with an roc of 0.639 . it was also more accurate than chronological age with an roc of 0.649 , illustrating the importance of illness over age . figure 2 compares the performance of these indices against each other . in all of these comparisons , not only was the cross - validated improvement in accuracy achieved by the mm index statistically significant , but the magnitude of the improvement was large enough to potentially explain away small or medium treatment effects . in the study of intensive care patients ( discussed above in the comparison of the mm index to physiological markers ) , we also compared the performance of the mm index to comorbidity categories within the elixhauser list , immunosuppressant medication use , and age . in contrast , the roc for immunosuppressant medication use was 0.59 ; for age it was 0.60 ; for elixhauser comorbidities it was 0.69 ; and for all combined variables ( including physiological markers ) it was 0.80 . as in previous studies , these differences were all cross - validated and statistically significant . the fact that the mm index , which scores each diagnosis , was more accurate than the elixhauser s categories of comorbidities suggests that grouping diagnoses into broad diagnostic categories reduces the accuracy of predictions . to illustrate the application of the mm index to specific cases , we use a case from a recent analysis of the prognosis of nursing home residents . the resident was 81 years old with 10 diagnoses during the last hospital admission ( see table 1 ) . the likelihood ratio of each diagnosis was looked up in the online table from the george mason university dataverse.68 for one diagnosis , chronic airway obstruction , not copd , and not elsewhere classified , the table does not provide any information and therefore this diagnosis was ignored and scored with a likelihood ratio of 1 . the mm score , the product of all likelihood ratios , was calculated as 45.07 . in this database , the prior odds was multiplied by the product of the likelihood ratios to obtain the posterior odds . the posterior odds can be expressed as a probability by dividing the posterior odds by one plus the odds . this patient s diagnoses has resulted in a probability of 0.88 for dying in the next six months . likelihood ratios above 1 indicate diagnoses that increased the odds of mortality . based on these data , the main reason for the high estimate of mortality pertained to the following : ( 1 ) lung cancer , ( 2 ) anorexia , and ( 3 ) cachexia . each more than doubled the risk of mortality . kidney disease also contributed to the high probability of mortality , but to a lesser extent . the following example can also demonstrate how the mmm variant of the mm index can be used . a patient presents with the worst disease in the circulatory system ( i.e. , second hospitalization for cardiogenic shock ) and has two diseases within the digestive system ( i.e. , first hospitalization with perforation of intestine and first hospitalization for malignant neoplasm of stomach , not otherwise specified ) . for simplicity , assume that this patient has no other diseases within other body systems . the maximum progression within circulatory disease is 1 , and the maximum progression within digestive diseases is 0.2 ( the maximum of 0.1 and 0.2 ) . for all other categories , there is no progression and they are scored 0 . the overall probability of death for the patient is then calculated as follows : mmm=1-(1 - 1.00.74)(1 - 0.200.81)=0.78 note that in this method of scoring , any diagnosis with elevated risk , no matter how benign , always increases the probability of mortality . to facilitate the use of the mm index in different ehr systems , appendix a contains an sql code that will facilitate the estimation of the parameters of the index for a specific cohort of patients within a data warehouse . while we have experience with icd-9 codes , a similar sql can be run for icd-10 , thereby allowing the ehr to adjust for changes in the coding procedures . in addition , a similar sql code can be run for procedures , exposure to medications , and categorized physiological measures , as well as decades of age , thereby allowing the prognostic index to reflect more than diagnoses . this paper reviews the recent methodological research on the performance and application of the mm index . one finding of this review was that mm index had higher roc than various physiological measures of prognosis , including ejection fraction for heart failure , hba1c levels for diabetic patients , and 13 physiological measures for patients in intensive care units . clinicians may prefer relying on physiological measures for any of the following reasons : many are concerned with coding errors in reporting patient diagnoses.69 however , given the accuracy of the results reported in this paper , coding errors are not extensive enough to significantly reduce the accuracy of diagnosis - based indices.some clinicians are concerned with reports that diagnoses in administrative data are less accurate than physiological markers when predicting prognosis , e.g. , brinkman et al.70 these studies do not contradict the data presented in this paper . as reviewed , when diagnoses are not grouped into broad categories , the results are radically different and worse than when each diagnosis is scored.some clinicians prefer to use physiological markers because they often use these markers in patient management . clearly , abnormal physiological markers raise the chances of mortality , but physiological markers are often transient and return to normal with good care . there is no reason to believe that these variations affect long - term mortality six months later . many are concerned with coding errors in reporting patient diagnoses.69 however , given the accuracy of the results reported in this paper , coding errors are not extensive enough to significantly reduce the accuracy of diagnosis - based indices . some clinicians are concerned with reports that diagnoses in administrative data are less accurate than physiological markers when predicting prognosis , e.g. , brinkman et al.70 these studies do not contradict the data presented in this paper . as reviewed , when diagnoses are not grouped into broad categories , the results are radically different and worse than when each diagnosis is scored . some clinicians prefer to use physiological markers because they often use these markers in patient management . clearly , abnormal physiological markers raise the chances of mortality , but physiological markers are often transient and return to normal with good care . there is no reason to believe that these variations affect long - term mortality six months later . a major conclusion of this paper was that mm index is more accurate than existing diagnosis - based indices , such as variants of charlson and elixhauser indices . to the best of our knowledge , the mm index is the first index that scores each disease separately rather than grouping similar diagnostic codes into broad categories . the observed improved accuracy of the mm index may be due to this feature of its scoring . the comprehensive inclusion of thousands of comorbidities in the mm index makes its use within clinical settings more difficult . to address use in clinical settings , the mmm variant still scores each disease but classifies these diseases by body system after scoring and selects the maximum ( worst ) score within each body system . this organization of diagnoses in various body systems allows clinicians to think through the prognosis of their patients more intuitively and without access to a computer . the mmm index is also helpful for conducting a propensity - matched comparative effectiveness study , where cases and controls must have similar scores across different body systems . matching on scores ranges in the body systems may be more practical than matching on separate diagnoses within the body system . use of the mm index is more practical now that many clinics have access to ehrs . these computers have access to the patients diagnostic history , can score patient s prognosis , and can explain the top two or three reasons for the predicted prognosis . then , the use of mm index in a clinical setting will be akin to use of any laboratory test , where the results are available but details of how the results were obtained are masked . the appendix to this paper ( as well as cielo repository ) provides an sql code that can be used to estimate the parameters of the mm index from data within warehouses or ehr systems . this code allows investigators to estimate the parameters of the mm index for the population that they are working with . there are a number of ways that the mm index can be further improved , including the examination of interaction among diseases and removal of confounding in estimates of likelihood ratios . in addition , the mm index may be improved if medications , physiological markers , or procedures were used to predict prognosis . the use of the mm index , as well as other prognostic indices , is fraught with difficulties when personalizing data for one patient . no patient is the average patient , since patients are likely to experience a combination of comorbidities that may radically differ from the average patient . furthermore , a discussion of mortality with patients may be understood differently if it is framed in terms of survival as opposed to mortality . probabilities may be misunderstood , and patients may prefer to know expected survival days as opposed to the probability of mortality in six months . obviously , any discussion of prognosis with patients requires empathetic communication on the part of clinicians . these and other limitations continue to frustrate efforts to make data on prognostic information available to patients and their families . additional research is needed to clarify how to best communicate prognostic information to individual patients . the mm and mmm indices can be used easily in policy analysis , decision support , and program evaluation . in these uses , the index enables assessment of comparative effectiveness of treatment . because these indices are more accurate than existing comprehensive diagnosis - based indices , we recommend their use despite the complexity of the underlying scoring procedures .

limbal relaxing incisions ( lris ) are considered a relatively safe procedure with rapid stabilization and absence of infectious complications . do we need to readdress this last impression ? we report a case of nocardia endophthalmitis associated with an exudate at the site of an lri in a patient who underwent routine cataract surgery . this case , to the best of our knowledge , is the first report of its kind , stressing the need for a cautious approach to the adoption of this method of astigmatic correction . background : the multimorbidity ( mm ) index predicts the prognosis of patients from their diagnostic history . in contrast to existing approaches with broad diagnostic categories , it treats each diagnosis as a separate independent variable using individual international classification of disease , revision 9 ( icd-9 ) codes.objective:this paper describes the mm index , reviews the published data on its accuracy , and provides procedures for implementing the index within electronic health record ( ehr ) systems . methods : the mm index was tested on various patient populations by using data from the united states department of veterans affairs data warehouse and claims data within the healthcare cost and utilization project of the agency for health care research and quality.results:in cross - validated studies , the mm index outperformed prognostic indices based on physiological markers , such as cd4 cell counts in hiv / aids , hbalc levels in diabetes , ejection fractions in heart failure , or the 13 physiological markers commonly used for patients in intensive care units . when predicting the prognosis of nursing home patients by using the cross - validated area under a receiver operating characteristic ( roc ) curve , the mm index was 15 percent outperformed the quan variant of the charlson index , 27 percent more accurate than the deyo variant of the charlson index , and 22 percent more accurate than the von walraven variant of the elixhauser index . for patients in intensive care units , the mm index was 13 percent outperformed the cross - validated area under roc associated with elixhauser s categories . the mm index also demonstrated greater accuracy than a number of commercially available measures of illness severity ; including a fivefold greater accuracy than the all patient refined diagnosis - related groups and a threefold greater accuracy than all payer severity - adjusted diagnosis - related groups.conclusion:the mm index is statistically more accurate than many existing measures of prognosis . the magnitude of improvement is large and may lead to a clinically meaningful difference in patient care . given the large improvements in accuracy , the use of the mm index for policy and comparative effectiveness analysis is recommended .

the choroid is a vessel - rich structure located between the lamina fusca of the sclera and the retinal pigment epithelium . choroidal thickness ( ct ) is thought to indicate the amount of choroidal vascularization and varies by age , sex , axial length ( axl ) , refractive status , and circadian rhythm.1,2 the choroid is reported to play a role in many vision - threatening diseases such as choroidal neovascularization , polypoidal choroidal vasculopathy , central serous chorioretinopathy , and chorioretinal atrophy associated with high myopia.36 thus , choroidal structural alterations are increasingly evaluated in scientific studies to understand the pathophysiology of these critical diseases . phacoemulsification surgery is the most frequently performed eye surgery , especially at advanced ages , and is generally associated with good visual outcomes . however , cataract surgery is known to have adverse effects on the retina , such as progression in diabetic retinopathy and pseudophakic cystoid macular edema , and the pathogenesis of such conditions is not yet clarified.7,8 also , epidemiological studies show that cataract surgery is associated with the onset of age - related macular degeneration ( amd ) , although the association is still controversial.9 the development of amd may be induced after cataract surgery due to reasons such as inflammatory reactions associated with cataract surgery , increased free radicals after surgery , the release of growth factors and prostaglandins , and increased light exposure during surgery.1014 amd is a serious disease , threatening vision due to neovascularizations arising from the choroid . an increment in the retinal thickness was reported after cataract surgery even in healthy eyes with no detected morphological impairment using spectral domain optical coherence tomography ( sd - oct).15 given the close link between the retina and choroid , affected change in ct might also be expected after cataract surgery . evaluation of choroid has been restricted to ultrasonography and angiography previously , while the choroid can be examined in detail with sd - oct . sd - oct provides detailed histological sectional images of the retina in vivo and provides useful information for the diagnosis , evaluation of the treatment options , and follow - up of several retinal diseases.16 the present study aims to examine the possible alterations in the ct after uncomplicated phacoemulsification surgery using sd - oct and to investigate the potential mechanisms that may cause such changes . this prospective study was conducted at the department of ophthalmology , faculty of medicine , bozok university , yozgat , turkey . the study was approved by the bozok university ethical committee and was performed in accordance with the ethical principles described in the declaration of helsinki . all subjects enrolled in the study agreed to participate , met the inclusion criteria , and signed an informed consent agreement before any procedures were performed . patients with diabetes mellitus , hypertension , dyslipidemia , any known systemic diseases , uveitis , glaucoma , retinal vein / branch occlusion , or other vascular and inflammatory retinal diseases , and previous eye surgery were excluded from the study due to the effect on ct . cataract hardness of the patients was evaluated and standardized based on the lens opacities classification system ( locs ) iii staging system,17 and patients with severe cataracts that might prevent a reliable sd - oct measurement and poor fixation were excluded from the study . , fort worth , tx , usa ) was used in all surgeries , and same hydrophobic acrylic intraocular lens ( acriva ud 613 ; vsy biotechnology , istanbul , turkey ) was implanted in all cases . the patients were administered topical moxifloxacin for 1 week and topical prednisolone for 4 weeks after the surgery . all patients underwent a detailed ophthalmologic examination including visual acuity measurement , anterior segment biomicroscopy , dilated fundus examination , and intraocular pressure ( iop ) measurement ( applanation tonometry ) preoperatively and 1 month postoperatively . furthermore , axl measurement using optical biometry ( lenstar ls 900 ; haag - streit ag , koeniz , switzerland ) was performed in all cases , preoperatively . sd - oct measurements were obtained in the morning ( between 9 am and 11 am ) in order to avoid the effect of diurnal fluctuations . changes in the ct after surgery and correlation of this change with age , axl , ept , preoperative iop , and iop change were evaluated . choroidal imaging was performed using rtvue-100 sd - oct system ( optovue inc , fremont , ca , usa ) . the depth resolution of the system is 5 m in tissue . for ct measurement , the patient s head and chin were properly positioned , then the patient was asked to maintain fixation on the internal fixation light , and a reverse image was obtained from the retina . the image was automatically inverted so that the chorioretinal interface was adjacent to the zero delay . the retina cross - line was used as the scanning pattern ; this mode provides two orthogonally oriented 6 mm lines consisting of 1024 a - scans , perpendicular to each other . sections passing through the fovea vertically and 1.5 and 3.0 mm nasal and temporal to the fovea were used for the measurements . the choroidal margins were taken as the distance between the outer margin of hyperreflective retinal pigment epithelium and the internal side of the sclera . only images with a signal strength 45 were taken . ct measurements were performed manually by two different physicians ( sab and hab ) , who were unaware of each other . these measurements were averaged ; the difference between the measurements of the physicians was 10% of the average . chicago , il , usa ) . the test was used to make comparisons between the categorical data . the shapiro since the data were distributed normally , the paired - samples t - test was used for the comparison between preoperative and postoperative measurements . potential parameters that might be associated with ct were evaluated using pearson s correlation analysis and regression analysis . evaluations were made at a 95% confidence interval , and a p - value < 0.05 was considered statistically significant . choroidal imaging was performed using rtvue-100 sd - oct system ( optovue inc , fremont , ca , usa ) . the depth resolution of the system is 5 m in tissue . for ct measurement , the patient s head and chin were properly positioned , then the patient was asked to maintain fixation on the internal fixation light , and a reverse image was obtained from the retina . the image was automatically inverted so that the chorioretinal interface was adjacent to the zero delay . the retina cross - line was used as the scanning pattern ; this mode provides two orthogonally oriented 6 mm lines consisting of 1024 a - scans , perpendicular to each other . sections passing through the fovea vertically and 1.5 and 3.0 mm nasal and temporal to the fovea were used for the measurements . the choroidal margins were taken as the distance between the outer margin of hyperreflective retinal pigment epithelium and the internal side of the sclera . only images with a signal strength 45 were taken . ct measurements were performed manually by two different physicians ( sab and hab ) , who were unaware of each other . these measurements were averaged ; the difference between the measurements of the physicians was 10% of the average . all data were analyzed using spss software ( version 16.0 , spss inc . , chicago , il , usa ) . the test was used to make comparisons between the categorical data . the shapiro wilk test was used to determine whether the data were distributed normally . since the data were distributed normally , the paired - samples t - test was used for the comparison between preoperative and postoperative measurements . potential parameters that might be associated with ct were evaluated using pearson s correlation analysis and regression analysis . evaluations were made at a 95% confidence interval , and a p - value < 0.05 was considered statistically significant . thirty - eight eyes of 38 patients ( 20 women and 18 men ) were included . there was only one patient with grade iv cataract hardness ; the rest of the patients had grade ii the mean duration of the operation was 14.783.47 minutes , and the mean ept was 6.373.47 seconds . the mean subfoveal ct was 247.1064.92 m before surgery ( figure 1 ) and 267.8474.1 m at 1 month postoperative follow - up ( p=0.002 ; figure 2 ) . a significant increase in the ct was observed in all regions ( p<0.05 for all ) ; such an increase was especially evident in the nasal and subfoveal regions . the iop was significantly lower at the 1-month follow - up evaluation than preoperatively ( 16.144.94 mmhg vs 13.914.86 mmhg ; p<0.001 ) . when the potential factors affecting the change in the ct were analyzed , the change in the ct was correlated with a change in iop for all sectors ; no significant correlation was observed between ct changes and age , axl , ept , or preoperative iop levels ( tables 2 and 3 ) . it has been suggested that cataract surgery may be associated with the onset of amd . the beaver dam and blue mountains groups reported an increased incidence of amd with cataract surgery.18,19 in contrast , in the age - related eye disease study , there was no significant increase in the incidence of amd after cataract surgery.20 although the fundoscopic appearance of the retina does not change with phacoemulsification in most eyes undergoing cataract surgery , subclinical changes in the macula and choroid have been reported after phacoemulsification surgery , as indicated by sd - oct sectional imaging of the retina.21 the effects of such subclinical changes are not yet fully understood . sd - oct imagery highlights the importance of ct changes in diseases of the choroid and retina , such as diabetic retinopathy , amd , and central serous chorioretinopathy.5,6,22,23 pierru et al24 examined the subfoveal ct after cataract surgery ; they reported that the ct began to increase postoperatively on day 7 and reached its highest value 1 month postoperatively . the follow - up exam at 3 months showed a decrease in ct.24 noda et al21 reported that subfoveal ct increased as indicated in the follow - up examination 1 month after surgery ; however , subfoveal ct did not return to preoperative values as indicated in the 6-month follow - up examination . another study evaluating the ct after cataract surgery found no significant changes in ct values 1 week and 1 month after surgery;25 however , the study was conducted with a limited number of participants ( 14 patients ) . in the present study we administered anti - inflammatory ( prednisolone ) eye drops to our patients for 1 month following surgery . anti - inflammatory therapies are known to reduce postoperative inflammation and suppress the inflammatory response in the retina.26 anti - inflammatory therapies may also reduce the postoperative choroidal response . we administered postoperative anti - inflammatory therapy to all of our patients as part of our standard treatment protocol in this study to evaluate the actual postoperative choroidal response . the mechanism through which cataract surgery increases inflammation in the retina and choroid has yet to be fully explained . it has been suggested that the ct may increase due to proinflammatory cytokines and prostaglandins that are believed to increase after cataract surgery.27,28 it is known that surgical trauma induces aqueous humor prostaglandin release and that these prostaglandins impair the blood aqueous barrier is impaired , other inflammatory mediators , such as endotoxin , cytokines , and immune complexes , accumulate in the aqueous humor . these inflammatory mediators pass through the vitreous to the retina , leading to an impaired inner and outer blood retinal barrier.29 the inflammatory response in the anterior segment , which develops secondary to cataract surgery , may thereby also cause inflammatory cascades in the posterior segment . however , these mechanisms can not explain why the increased choroidal blood supply continues for up to 6 months after cataract surgery . another mechanism suggested for inflammation induced by surgical trauma is increased gene expression that simultaneously induces inflammatory response in all structures of the eye . in a mouse model , extracapsular lens extraction was found to increase protein secretion in the retina and choroid through proinflammatory gene expression . an increase was observed in the expression of genes related to chemokines such as ccl-2 and sdf-1 , which can impair the blood retinal barrier in the retina , retinal pigment epithelium , and choroid in operated eyes , as well as growth factors such as fibroblast growth factors and vascular endothelial growth factors . the same study also established that il-1 , a proinflammatory cytokine , increased in the ganglion cell layer , inner nuclear layer , and choroid in operated eyes.28 another animal study involving monkeys showed that cataract surgery destroyed the outer blood retinal barrier and increased macular thickness.29 it has been shown that even uncomplicated phacoemulsification has effects on retina , in general as temporary thickening of the central retina . this effect could be attributed to factors such as ultrasound power used during surgery or the intraoperative iop fluctuations caused by surge or the intraoperative photostress due to microscope light.30 when we evaluated our data on the correlation between ept and ct , we did not find any correlation . though the cataract hardness of the patients in our study ranged from grade ii to iv based on locs iii , patients with severe cataracts that might prevent a reliable sd - oct measurement were excluded , and there was only one patient with grade iv cataract hardness . since the patients expecting high ultrasonic energy due to hard cataracts were excluded , our patient group was homogeneous in terms of cataract hardness , and this might prevent detection of any possible correlation between ept and ct . on the other hand , ching et al31 showed thicker preoperative retinal thickness than those in the early postoperative period , and the authors suggested that possible measurement errors in preoperative evaluation might be the reason of their results . so , all these factors might also have an influence on ct , and while interpreting our results , these factors should also be kept in mind . iop decreases after cataract surgery.32 to understand the reasons for iop decrease after phacoemulsification , many factors should be taken into account . by removing the thick lens , blockage of aqueous outflow with lens is prevented , the angle of anterior chamber opens , aqueous outflow improves , and consequently iop decreases . a study by ohsugi et al33 with 100 patients reported a negative correlation between change in ct and change in iop in the postoperative early period , supporting the present study ; however , they did not establish any correlation between ct change and iop for many regions in subsequent periods . although their recent report showed a significant correlation between change in ct and change in iop in the early postoperative period , we believe that this is worth confirming in a further study . also , in our study , the same experienced surgeon performed all surgeries and the same intraocular lens was implanted in all cases , whereas in the study by ohsugi et al,33 seven surgeons performed the surgeries and uniformity of intraocular lens was not mentioned , so these might have limited standardization of the surgical technique in their preliminary report . the increase in ct may be due to increased ocular perfusion pressure caused by reduced iop in the early period after cataract surgery . the driving force for ocular blood flow is the ocular perfusion pressure , defined as ocular arterial pressure minus iop.34 animal studies showed that induction of ocular hypertension resulted in decreased blood flow through the choroid , while retinal blood flow remained at normal levels , suggesting a marked difference in autoregulatory capacity between the choroidal and retinal vascular beds.35 it was also demonstrated that in vivo subfoveal ct as measured by enhanced depth imaging - oct was significantly associated with ocular perfusion pressure , suggesting that subfoveal ct may be indirectly indicative of subfoveal ocular perfusion status.36 since there is an inverse relation between iop and ocular perfusion pressure , the increase in ct may be due to increased ocular perfusion pressure caused by reduced iop in the early period after cataract surgery in our study . in the future , ct relationship may be understood upon evaluation of changes in ct after glaucoma surgery , which greatly influences iop . in this study , we did not find any correlation between the axl and age with ct ; however , the included subjects were older patients within nearly the same age range , and the patients having myopic fundus were not included in the study . thus , these might have limited our results on the correlation between the axl and age with ct . cataract surgery causes an increase in ct , which is correlated with a change in iop in the early stages . this is especially important for understanding the pathogenesis of diseases associated with choroidal changes , such as cystoid macular edema , which increase in prevalence following cataract surgery . while augmentation cystoplasty has seen its indications decrease with the advent of onabotulinum toxin a and neuromodulation , there remains a role for this procedure in the 21st century ( 1 ) . analysis from the nationwide inpatient sample ( nis ) reveals that augmentation cystoplasty appears to be widely used in the surgical management of neurogenic bladder in patients with spina bifida ( 2 ) . data from the pediatric health information system ( phis ) database demonstrates no change in augmentation rates between 1999 and 2004 , finding 665 patients who underwent augmentation cystoplasty over this 5-year period ( 3 ) . a recent study by uc san francisco demonstrated a decrease in the number of augmentations by 25% in the 2000s ( 4 ) . institutional case series have examined complications of bladder reconstruction , suggesting it continues to have notable morbidity . retrospectively analyzed 25 pediatric patients who underwent augmentation cystoplasty ( 5 ) and bladder substitution ( 6 ) between 1999 and 2007 , finding major complications including lower urinary tract calculi ( 39% ) , stricture or insufficiency of the continent vesicostomy ( 28% ) , and intestinal obstruction ( 9% ) ( 5 ) . approximately one - third of patients who have undergone enterocystoplasty experience postoperative complications ( 6 , 7 ) . these morbidities are secondary to a multitude of factors including surgical technique , inherent properties of the bowel segment , and patient compliance with maintenance regimens ( 8) . we sought to determine , on a national scale , the readmission rates at 30 , 60 and 90 days after initial surgery , whether they have improved over the 7-year period , and to determine the influence of hospital and patient - specific variables with estimated costs of readmission . this was a retrospective cohort analysis of the phis , a national administrative database containing combined hospitalization data from 43 tertiary care pediatric hospitals in the united states . this study was reviewed and approved by the uc san diego human research protections program institutional review board ( irb ) with an informed consent waiver . patients were followed across multiple hospital admissions to the same phis hospital using unique numerical patient identifiers . patient privacy and confidentiality are protected and this study conforms to the ethical guidelines of the 1975 declaration of helsinki . inpatient data , emergency room visits , observation , and ambulatory surgery / medicine visits are all included . some hospitals contribute varying amounts of data , for example , only inpatient visits versus inpatient , ambulatory , emergency room , and observation visits . each participating institution and an independent data management group review the data for accuracy each quarter . data is only accepted into phis if classified errors occur in less than 2% of the quarterly data . thirty - seven of the 43 hospitals had complete inpatient information during the study period and were included in the analysis . we identified patients 0 - 17 years of age , from 2004 - 2010 , undergoing bladder reconstruction using icd-9 procedure codes : 56.51 ( formation cutaneous uretero - ileostomy ) , 57.87 ( reconstruction of urinary bladder ) , and 57.88 ( other anastomosis of bladder ) ; patients undergoing repair of bladder exstrophy ( icd9 57.86 ) were excluded . descriptive statistics were used to characterize patient demographics , prevalence of surgeries , and readmission rates . surgery prevalence was examined using linear regression and readmission rates were compared using the chi test . total charges were compared between those who required readmission and those who did not , using the mann - whitney u - test . costs are recorded in the phis database using a cost - to - charges ratio ( rcc - based ) for each hospital , which standardizes costs nationally taking into account variability in pricing and costs regionally . although the rcc method is a good approximation for true costs , these costs are estimates and as such are only used in this analysis to compare between groups , rather than reporting them as a true exact cost amount . ninety - day readmission rates were compared between hospitals after adjusting for hospital volume of augmentation surgery ( number of readmits / total number of augment surgeries ) . gee ( generalized estimating equation ) regression was used to evaluate the influence of independent variables on 90-day readmission risk while adjusting for patient clustering within hospitals . covariates entered into the original model included age , gender , race , insurance ( public / private ) , complex chronic condition ( which comprises a broad array of icd 9 codes for congenital anomalies ) ( 9 ) , length of stay ( < = 7 , > 7 days ) , year of surgery , days admitted prior to surgery to accommodate for bowel preps ( 0 or 1 ) and all 2-term interactions . only variables that remained significant in multivariate analysis remained in the final models . significance for testing was set a priori at p < 0.05 , and stata ver . this was a retrospective cohort analysis of the phis , a national administrative database containing combined hospitalization data from 43 tertiary care pediatric hospitals in the united states . this study was reviewed and approved by the uc san diego human research protections program institutional review board ( irb ) with an informed consent waiver . patients were followed across multiple hospital admissions to the same phis hospital using unique numerical patient identifiers . patient privacy and confidentiality are protected and this study conforms to the ethical guidelines of the 1975 declaration of helsinki . inpatient data , emergency room visits , observation , and ambulatory surgery / medicine visits are all included . some hospitals contribute varying amounts of data , for example , only inpatient visits versus inpatient , ambulatory , emergency room , and observation visits . each participating institution and an independent data management group review the data for accuracy each quarter . data is only accepted into phis if classified errors occur in less than 2% of the quarterly data . thirty - seven of the 43 hospitals had complete inpatient information during the study period and were included in the analysis . we identified patients 0 - 17 years of age , from 2004 - 2010 , undergoing bladder reconstruction using icd-9 procedure codes : 56.51 ( formation cutaneous uretero - ileostomy ) , 57.87 ( reconstruction of urinary bladder ) , and 57.88 ( other anastomosis of bladder ) ; patients undergoing repair of bladder exstrophy ( icd9 57.86 ) were excluded . descriptive statistics were used to characterize patient demographics , prevalence of surgeries , and readmission rates . surgery prevalence was examined using linear regression and readmission rates were compared using the chi test . total charges were compared between those who required readmission and those who did not , using the mann - whitney u - test . costs are recorded in the phis database using a cost - to - charges ratio ( rcc - based ) for each hospital , which standardizes costs nationally taking into account variability in pricing and costs regionally . although the rcc method is a good approximation for true costs , these costs are estimates and as such are only used in this analysis to compare between groups , rather than reporting them as a true exact cost amount . ninety - day readmission rates were compared between hospitals after adjusting for hospital volume of augmentation surgery ( number of readmits / total number of augment surgeries ) . gee ( generalized estimating equation ) regression was used to evaluate the influence of independent variables on 90-day readmission risk while adjusting for patient clustering within hospitals . covariates entered into the original model included age , gender , race , insurance ( public / private ) , complex chronic condition ( which comprises a broad array of icd 9 codes for congenital anomalies ) ( 9 ) , length of stay ( < = 7 , > 7 days ) , year of surgery , days admitted prior to surgery to accommodate for bowel preps ( 0 or 1 ) and all 2-term interactions . only variables that remained significant in multivariate analysis remained in the final models . significance for testing was set a priori at p < 0.05 , and stata ver . , there has been no change in the prevalence of bladder reconstruction surgeries ( r : 0.191 , coefficient : -4.71 , 95% ci : -15.9 to 6.4 , p = 0.327 ; figure 1 ) . there was no change in 30-day ( ranging from 14% to 20% p = 0.272 ) , 60-day ( 19% to 24% , p = 0.788 ) or 90-day readmission rates over time ( 23% to 27% , p = 0.924 ) ( table 2 ) . despite surgical volume adjustment , the majority of hospitals had similar 90-day readmission rates ( figure 2 ) . from a total of 37 hospitals , two had significantly higher readmission rates / volume and three had significantly lower readmission rates / volume than the mean . the median cost for those patients requiring readmission was 1.76-fold higher than that of patients who were not readmitted based on rcc ( p < 0.001 ) . 2 hospitals had significantly higher readmission rate than the mean ( * ) , and 3 had significantly lower readmission rates ( ^ ) . on multivariate analysis , a significant interaction was seen between gender and complex chronic condition ( ccc ) and as such , additional models were generated after splitting by gender : initial los > 7 days had 2-fold higher odds of readmission than < = 7 ( males or 1.647 , p = 0.001 ; females or 2.18 , p < 0.001 ) . within males , having a ccc was significantly associated with 90-day readmission ( or 1.898 , p = 0.001 ) ; ccc was not an independent predictor in females ( p = 0.147 ) . between 2004 and 2010 , there was no significant change in the prevalence in bladder reconstruction in the pediatric population , nor was there any change in the rate of readmission of these post - surgical patients at 30 , 60 , or 90 days . a recent study by schlomer et al . shows a 25% decrease in bladder augmentation over the last decade ( 4 ) . however , the critical issue of this study is the need for early postoperative readmission , which has been stable and alarmingly high . beyond the direct morbidity to the patient , there is also a tremendous cost to the health care system with a 1.7-fold increase over patients who are not readmitted . the 90-day admission rate of 23 - 27% is comparable to the 30% readmission rate seen following either adult kidney or liver transplantation , which speaks to the severity of the condition and surgery ( 10 , 11 ) . following urinary diversion in adults , the 90-day readmission rate has also been reported to be near 30% as well ( 12 ) . when counseling families and practitioners , the significant likelihood of readmission should not be underestimated . single institutional studies have been able to identify long - term morbidity following reconstruction . in adults , welk et al . reported the rate of additional urological surgery after adult enterocystoplasty , finding 40% of patients required subsequent urological surgery in 243 patients , with a median follow up of 7.8 years ( 13 ) . similar to adults , there is impressive morbidity after total bladder substitution in the pediatric population . defoor et al . retrospectively analyzed a population of 26 patients , median age 8.9 years , with median follow up of 8 years , who underwent neobladder creation , finding complications including reservoir stones ( 8 patients ) , febrile urinary tract infections ( 8 patients ) , metabolic acidosis ( 6 patients ) , small bowel obstruction ( 4 patients ) , bladder perforation ( 4 patients ) , and pelvic lymphocele ( 1 patient ) ( 14 ) . although the short- to long - term complications following pediatric bladder augmentation / reconstruction have been enumerated , the likelihood of early readmission following initial surgery has not been studied from a population - based perspective . despite including 37 phis hospitals nationwide in our analysis with a diverse volume of bladder augmentation caseloads , our analysis found no appreciable difference in postoperative readmission rates among hospitals . this could be secondary to several reasons , including the consistency of pediatric urologists at the majority of phis hospitals , the essential high morbidity of the surgery itself and its predisposing condition , or even the likelihood that an increased presence of patients with cccs at high volume centers may dilute the otherwise improved outcomes in the overall patient population . there has been recent discussion within the adult genitourinary surgical community focusing on the regionalization of radical prostatectomy to high volume centers , secondary to the belief this would provide patients with improved outcomes . analysis compiled from the nis concluded that low volume institutions experienced inferior outcomes relative to the highest volume centers , irrespective of approach for radical prostatectomy , supposedly demonstrating the importance of accounting for hospital volume when examining the benefit of a surgical technique ( 15 ) . however , in the present study cohort when adjusted for hospital volume , there is a similar readmission rate among all institutions . in 2011 , berry et al . used the phis database to characterize hospital readmissions between 2003 and 2008 , finding 21.8% of patients experienced at least 1 readmission within 365 days of a prior admission ; 2.9% experienced 4 or more readmissions within 365 days , accounting for 18.8% of overall admissions ( 16 ) . of this 2.9% , 28.5% ( 2633 ) were re - hospitalized for a problem in the same organ system across all admissions during the interval . a retrospective analysis examined readmission among 1,083 patients hospitalized between 2006 and 2008 who were part of a structured , pediatric complex - care clinical program within 4 children s hospitals . notably , almost half ( 47.1% ) of all complex - care clinical program patient admissions were related primarily to a surgical operation or major procedure ( 17 ) . in our analysis , multivariate regression found males with cccs were at increased risk of postoperative readmission ; this is supported by previous data which has already identified the use of cccs in models describing pediatric readmission rates ( 18 , 19 ) . somewhat surprising was our finding that ccc was an independent predictor of readmission in males but not females . this could be secondary to a yet unidentified gap in the care of male patients . however , 83.5% of our cohort had a ccc , with 50% reporting a neuromuscular condition . as a result , the attempted use of cccs for determining readmission risk is not ideal but was an attempt to find some way of quantifying risk . we attempted to find other predictors of readmission including specific conditions such as obesity but only initial los > 7 days during the initial surgery yielded a 2-fold increase in likelihood of readmission . rice - townsend et al . utilized a retrospective analysis of the phis database to identify disease severity among patients as the primary factor associated with readmission after treatment of appendicitis ( 20 ) . this factor was highly variable across hospital populations and significantly altered ranking of performance - based outliers . one of the limitations of our study is the inability to account for disease severity , going back to the lack of unified , descriptive data that is needed to better stratify and describe the cohort of pediatric patients undergoing bladder reconstruction . additional limitations include a lack of readmission data in situations where pediatric patients were re - hospitalized in non - phis hospitals or patients were readmitted to a different phis hospital . patients may have been readmitted for other reasons outside of the initial bladder reconstruction , thus overestimating our readmission rates . the lack of sufficient finite data to characterize the reasons for readmission is a significant drawback in administrative databases , but we believe that a definite trend is seen in this study . the ability to generalize our results might also be limited to freestanding children s hospitals and does not represent the national norm , although it is estimated that 25% of children s hospitalizations occur within children s hospitals ( national statistics on children , healthcare cost and utilization project , agency for healthcare research and quality , http://www.hcupnet.ahrq.gov/ ( accessed march 30 , 2013 ) . children treated at children s hospitals have a higher prevalence of cccs than children at other hospitals , and this could potentially affect our results ( 21 ) . in addition , our use of icd-9 coding to identify patients for inclusion is always subject to the quality of initial data collection and may unintentionally lead to inclusion of inappropriate or exclusion of appropriate patients . with health care reform initiatives looming , the future of medicine will likely include bundled payments for inpatient surgical and outpatient post - hospitalization care , ensuring accountability for patient outcomes and inpatient utilization among care providers ( 22 - 24 ) . pediatric bladder reconstruction is a significant endeavor with an essential likelihood for future admission at substantial cost . as evidenced in this study , readmission rates after pediatric bladder reconstruction have not improved over time . single institutional studies have been able to identify long - term morbidity following reconstruction . in adults , welk et al . reported the rate of additional urological surgery after adult enterocystoplasty , finding 40% of patients required subsequent urological surgery in 243 patients , with a median follow up of 7.8 years ( 13 ) . similar to adults , there is impressive morbidity after total bladder substitution in the pediatric population . defoor et al . retrospectively analyzed a population of 26 patients , median age 8.9 years , with median follow up of 8 years , who underwent neobladder creation , finding complications including reservoir stones ( 8 patients ) , febrile urinary tract infections ( 8 patients ) , metabolic acidosis ( 6 patients ) , small bowel obstruction ( 4 patients ) , bladder perforation ( 4 patients ) , and pelvic lymphocele ( 1 patient ) ( 14 ) . although the short- to long - term complications following pediatric bladder augmentation / reconstruction have been enumerated , the likelihood of early readmission following initial surgery has not been studied from a population - based perspective . despite including 37 phis hospitals nationwide in our analysis with a diverse volume of bladder augmentation caseloads , our analysis found no appreciable difference in postoperative readmission rates among hospitals . this could be secondary to several reasons , including the consistency of pediatric urologists at the majority of phis hospitals , the essential high morbidity of the surgery itself and its predisposing condition , or even the likelihood that an increased presence of patients with cccs at high volume centers may dilute the otherwise improved outcomes in the overall patient population . there has been recent discussion within the adult genitourinary surgical community focusing on the regionalization of radical prostatectomy to high volume centers , secondary to the belief this would provide patients with improved outcomes . analysis compiled from the nis concluded that low volume institutions experienced inferior outcomes relative to the highest volume centers , irrespective of approach for radical prostatectomy , supposedly demonstrating the importance of accounting for hospital volume when examining the benefit of a surgical technique ( 15 ) . however , in the present study cohort when adjusted for hospital volume , there is a similar readmission rate among all institutions . in 2011 , berry et al . used the phis database to characterize hospital readmissions between 2003 and 2008 , finding 21.8% of patients experienced at least 1 readmission within 365 days of a prior admission ; 2.9% experienced 4 or more readmissions within 365 days , accounting for 18.8% of overall admissions ( 16 ) . of this 2.9% , 28.5% ( 2633 ) were re - hospitalized for a problem in the same organ system across all admissions during the interval . a retrospective analysis examined readmission among 1,083 patients hospitalized between 2006 and 2008 who were part of a structured , pediatric complex - care clinical program within 4 children s hospitals . notably , almost half ( 47.1% ) of all complex - care clinical program patient admissions were related primarily to a surgical operation or major procedure ( 17 ) . in our analysis , multivariate regression found males with cccs were at increased risk of postoperative readmission ; this is supported by previous data which has already identified the use of cccs in models describing pediatric readmission rates ( 18 , 19 ) . somewhat surprising was our finding that ccc was an independent predictor of readmission in males but not females . this could be secondary to a yet unidentified gap in the care of male patients . however , 83.5% of our cohort had a ccc , with 50% reporting a neuromuscular condition . as a result , the attempted use of cccs for determining readmission risk is not ideal but was an attempt to find some way of quantifying risk . we attempted to find other predictors of readmission including specific conditions such as obesity but only initial los > 7 days during the initial surgery yielded a 2-fold increase in likelihood of readmission . rice - townsend et al . utilized a retrospective analysis of the phis database to identify disease severity among patients as the primary factor associated with readmission after treatment of appendicitis ( 20 ) . this factor was highly variable across hospital populations and significantly altered ranking of performance - based outliers . one of the limitations of our study is the inability to account for disease severity , going back to the lack of unified , descriptive data that is needed to better stratify and describe the cohort of pediatric patients undergoing bladder reconstruction . additional limitations include a lack of readmission data in situations where pediatric patients were re - hospitalized in non - phis hospitals or patients were readmitted to a different phis hospital patients may have been readmitted for other reasons outside of the initial bladder reconstruction , thus overestimating our readmission rates . the lack of sufficient finite data to characterize the reasons for readmission is a significant drawback in administrative databases , but we believe that a definite trend is seen in this study . the ability to generalize our results might also be limited to freestanding children s hospitals and does not represent the national norm , although it is estimated that 25% of children s hospitalizations occur within children s hospitals ( national statistics on children , healthcare cost and utilization project , agency for healthcare research and quality , http://www.hcupnet.ahrq.gov/ ( accessed march 30 , 2013 ) . children treated at children s hospitals have a higher prevalence of cccs than children at other hospitals , and this could potentially affect our results ( 21 ) . in addition , our use of icd-9 coding to identify patients for inclusion is always subject to the quality of initial data collection and may unintentionally lead to inclusion of inappropriate or exclusion of appropriate patients . with health care reform initiatives looming , the future of medicine will likely include bundled payments for inpatient surgical and outpatient post - hospitalization care , ensuring accountability for patient outcomes and inpatient utilization among care providers ( 22 - 24 ) . pediatric bladder reconstruction is a significant endeavor with an essential likelihood for future admission at substantial cost . as evidenced in this study ,

purposethe aim of the study was to analyze the effects of uneventful phacoemulsification surgery on choroidal thickness ( ct ) using spectral domain optical coherence tomography ( sd - oct).methodsin this prospective study , 38 eyes of 38 patients having phacoemulsification surgery were included . all patients underwent detailed ophthalmologic examination , including preoperative axial length ( axl ) measurement with optical biometry and intraocular pressure ( iop ) measurement preoperatively and 1 month postoperatively . the ct was measured perpendicularly at the fovea and 1.5 mm temporal , 3.0 mm temporal , 1.5 mm nasal , and 3.0 mm nasal using sd - oct preoperatively and 1 month postoperatively . changes in the ct after surgery and correlation of this change with age , axl , preoperative iop , and iop change were evaluated.resultsthere was a statistically significant increase in the ct at all regions evaluated . this increment was more prominent in the nasal and subfoveal regions . the iop decreased significantly 1 month after surgery ( 16.144.94 mmhg vs 13.914.86 mmhg ; p<0.001 ) . the change in iop was correlated with the ct changes at all regions , whereas age , axl , and preoperative iop had no significant correlations with the changes in ct.conclusionphacoemulsification surgery may cause significant increase in ct , which is correlated with surgery - induced iop change in the short term . long - term follow - up of eyes having phacoemulsification surgery may provide further insight into the effects of cataract surgery on the choroid . background : bladder reconstruction in the pediatric population is challenging for many reasons , including perioperative complications and readmissions.objectives:on a national scale , determine readmission rates at 30 , 60 and 90 days after bladder reconstruction in a pediatric population over a 7-year period , evaluating the influence of hospital and patient - specific variables.patients and methods : using the pediatric health information system database , we identified patients 0 - 17 years of age , from 2004 - 2010 , undergoing bladder reconstruction using icd-9 procedure codes . descriptive statistics characterized demographics , prevalence of surgeries , and readmission rates . surgery prevalence over time was examined using linear regression . readmission rates were compared using the chi2 test . regression was used to evaluate the influence of variables on readmission risk.results:we identified 1,985 patients for inclusion , of which 52.7% were female . median age was 9 years . there has been no change in the prevalence of bladder reconstruction surgeries ( p = 0.327 ) . there was no change in 30-day ( p = 0.272 ) , 60-day ( p = 0.788 ) or 90-day readmission rates ( p = 0.924 ) . despite surgical volume adjustment , 90-day readmission rates did not significantly vary among the majority of hospitals . initial los > 7 days ( p < 0.001 ) and complex chronic condition males ( p < 0.001 ) were significantly associated with 90-day readmission.conclusions:no improvement in readmission rates after pediatric bladder reconstruction was observed during the study period . nearly all centers have a similar readmission rate despite volume adjustment .

the prevalence of nephrolithiasis is 5% to 20%and varies with age , sex , race , and area of residence . nephrolithiasis is usually asymptomatic but the attacks can be painful and morbid , requiring an emergency medical visit and surgery . the focus on nephrolithiasis has shifted from these direct hazards to its effects , such as vascular events . nephrolithiasis and systemic diseases have been linked in several epidemiological studies , including coronary heart disease ( chd ) , hypertension , diabetes , atherosclerosis , and metabolic syndrome . ferraro et al demonstrated an association between kidney stones and an up to 1.48-fold higher risk of chd in prospective cohorts of women . atherosclerosis is the risk factor for chd and stroke , patients with nephrolithiasis might have a high stroke risk . domingos et al reported that patients with nephrolithiasis did not have a higher incidence of stroke than those without nephrolithiasis did . however , in their questionnaire - based study , adjustment for major confounders of stroke , such as hypertension , diabetes , and hyperlipidemia , was challenging in the absence of formal medical documentation . thus , a nationwide population database was used to assess the association of nephrolithiasis with stroke risks in a cohort study with a 13-year follow - up . the national health insurance ( nhi ) program was launched in taiwan on march 1 , 1995 , and covered > 99% of taiwan 's residents and was contracted with 97% of clinics and hospitals by the end of 2010 . this study utilized the longitudinal health insurance database 2000 ( lhid2000 ) claims data established by the national health research institute ( nhri ) , department of health , taiwan . lhid2000 contains the medical claims data of 1 million randomly sampled beneficiaries from among the enrollees of the nhi program between 1996 and 2010 . no statistically significant differences in the distribution of sex , age , or healthcare costs between cohorts in the lhid2000 and the enrollees were reported by the nhri . the international classification of diseases revision ninth clinical modification ( icd-9-cm ) was used for the diagnoses . the study was approved by the institutional review board of china medical university and hospital ( cmuh104-rec2115 ) . a retrospective cohort study was conducted to determine the association between renal stone ( rs ) ( icd-9-cm 592.0 : calculus of kidney , icd-9-cm 592.1 : calculus of ureter , icd-9-cm 592.9 : urinary calculus , icd-9-cm 594.1 : other calculus in bladder ) and stroke ( icd-9-cm 430 : subarachnoid hemorrhage , icd-9-cm 431 : intracerebral hemorrhage , icd-9-cm 432 : other and unspecified intracranial hemorrhage , icd-9-cm 433 : occlusion and stenosis of precerebral arteries , icd-9-cm 434 : occlusion of cerebral arteries , icd-9-cm 435 : transient cerebral ischemia , icd-9-cm 436 acute , but ill - defined , cerebrovascular disease , icd-9-cm 437 other and ill - defined cerebrovascular disease , icd-9-cm 438 : late effects of cerebrovascular disease ) . the claims data between1998 and 2010 were used to identify patients aged 20 years with a first diagnosis of rs . a comparison cohort was randomly selected from among the patients without rss during the same period , frequency matched 1:4 for age ( every 5 years ) , sex , comorbidities ( diabetes , hyperlipidemia , hypertension , coronary artery disease [ cad ] , congestive heart failure [ chf ] , and atrial fibrillation ) , and index year . patients with a stroke history and with missing information for age or sex were excluded from the data analysis . a follow - up for stroke occurrence was performed until the date of follow - up loss , withdrawal from the insurance system , or end of 2010 , whichever occurred first . the baseline comorbidities were diabetes ( icd-9-cm 250 ) , hyperlipidemia ( icd-9-cm 272 ) , hypertension ( icd-9-cm 401 to 405 ) , cad ( icd-9-cm 410 to 414 ) , chf ( icd-9-cm 398.91 , 402 , 404.01 , 404.03 , 404.10 , 404.11 , 404.13 , and 404.9 ) , and atrial fibrillation ( icd-9-cm 427.31 ) . surgical procedures for rs removal were based on icd-9-op codes ( 55.01 : nephrotomy , 55.03 : percutaneous nephrostomy without fragmentation , 55.04 : percutaneous nephrostomy with fragmentation , 56.0 : transurethral removal of obstruction from ureter and renal pelvis , 57.0 : transurethral clearance of bladder , 59.95 : ultrasonic fragmentation of urinary stones , 98.51 : extracorporeal shockwave lithotripsy of the kidney , ureter , and/or bladder ) . orthopedic surgeries of lower limbs were based on icd-9 op codes ( icd-9-cm 81.51 : total hip replacement , 81.52 : partial hip replacement , 81.53 : revision of hip replacement , 81.54 : total knee replacement ) . the distribution of demographic status and comorbidities , including age , sex , diabetes , hyperlipidemia , hypertension , cad , and chf were compared between the rs and non - rs cohorts . hazard ratios ( hrs ) and adjusted hrs ( ahrs ) were computed for stroke as a measure of the relative risk through the cox proportional hazards regression model . in addition , the cox model was used to estimate stroke hrs associated with the frequency of medical visits for stone removal and was compared with the patients with rss who received conservative medication . the stroke - free survival probability during the follow - up period was estimated using the kaplan meier method , and the difference in frequency of stone removal among patients with rss was examined using a log rank test . statistical analyses were performed using sas ( version 9.2 ; sas institute , cary , nc ) . the national health insurance ( nhi ) program was launched in taiwan on march 1 , 1995 , and covered > 99% of taiwan 's residents and was contracted with 97% of clinics and hospitals by the end of 2010 . this study utilized the longitudinal health insurance database 2000 ( lhid2000 ) claims data established by the national health research institute ( nhri ) , department of health , taiwan . lhid2000 contains the medical claims data of 1 million randomly sampled beneficiaries from among the enrollees of the nhi program between 1996 and 2010 . no statistically significant differences in the distribution of sex , age , or healthcare costs between cohorts in the lhid2000 and the enrollees were reported by the nhri . the international classification of diseases revision ninth clinical modification ( icd-9-cm ) was used for the diagnoses . the study was approved by the institutional review board of china medical university and hospital ( cmuh104-rec2115 ) . a retrospective cohort study was conducted to determine the association between renal stone ( rs ) ( icd-9-cm 592.0 : calculus of kidney , icd-9-cm 592.1 : calculus of ureter , icd-9-cm 592.9 : urinary calculus , icd-9-cm 594.1 : other calculus in bladder ) and stroke ( icd-9-cm 430 : subarachnoid hemorrhage , icd-9-cm 431 : intracerebral hemorrhage , icd-9-cm 432 : other and unspecified intracranial hemorrhage , icd-9-cm 433 : occlusion and stenosis of precerebral arteries , icd-9-cm 434 : occlusion of cerebral arteries , icd-9-cm 435 : transient cerebral ischemia , icd-9-cm 436 acute , but ill - defined , cerebrovascular disease , icd-9-cm 437 other and ill - defined cerebrovascular disease , icd-9-cm 438 : late effects of cerebrovascular disease ) . the claims data between1998 and 2010 were used to identify patients aged 20 years with a first diagnosis of rs . a comparison cohort was randomly selected from among the patients without rss during the same period , frequency matched 1:4 for age ( every 5 years ) , sex , comorbidities ( diabetes , hyperlipidemia , hypertension , coronary artery disease [ cad ] , congestive heart failure [ chf ] , and atrial fibrillation ) , and index year . patients with a stroke history and with missing information for age or sex were excluded from the data analysis . a follow - up for stroke occurrence was performed until the date of follow - up loss , withdrawal from the insurance system , or end of 2010 , whichever occurred first . the baseline comorbidities were diabetes ( icd-9-cm 250 ) , hyperlipidemia ( icd-9-cm 272 ) , hypertension ( icd-9-cm 401 to 405 ) , cad ( icd-9-cm 410 to 414 ) , chf ( icd-9-cm 398.91 , 402 , 404.01 , 404.03 , 404.10 , 404.11 , 404.13 , and 404.9 ) , and atrial fibrillation ( icd-9-cm 427.31 ) . surgical procedures for rs removal were based on icd-9-op codes ( 55.01 : nephrotomy , 55.03 : percutaneous nephrostomy without fragmentation , 55.04 : percutaneous nephrostomy with fragmentation , 56.0 : transurethral removal of obstruction from ureter and renal pelvis , 57.0 : transurethral clearance of bladder , 59.95 : ultrasonic fragmentation of urinary stones , 98.51 : extracorporeal shockwave lithotripsy of the kidney , ureter , and/or bladder ) . orthopedic surgeries of lower limbs were based on icd-9 op codes ( icd-9-cm 81.51 : total hip replacement , 81.52 : partial hip replacement , 81.53 : revision of hip replacement , 81.54 : total knee replacement ) . the distribution of demographic status and comorbidities , including age , sex , diabetes , hyperlipidemia , hypertension , cad , and chf were compared between the rs and non - rs cohorts . hazard ratios ( hrs ) and adjusted hrs ( ahrs ) were computed for stroke as a measure of the relative risk through the cox proportional hazards regression model . in addition , the cox model was used to estimate stroke hrs associated with the frequency of medical visits for stone removal and was compared with the patients with rss who received conservative medication . the stroke - free survival probability during the follow - up period was estimated using the kaplan meier method , and the difference in frequency of stone removal among patients with rss was examined using a log rank test . statistical analyses were performed using sas ( version 9.2 ; sas institute , cary , nc ) . in total , 53,659 patients with newly diagnosed rss and 236,107 patients without rss ( non - rs , comparison cohort ) between 1998 and 2010 were identified ( table 1 ) . the numbers of patients with follow - up loss were 1790 patients ( 3.34% ) in rs cohort and 7048 patients ( 3.29% ) in non - rs cohort . the median duration of follow - up was 6.78 years in the rs cohort ( 0.00313.0 years ) and 6.62 years in the non - rs cohort ( 0.00313.0 years ) . because the rs and non - rs groups were matched with respect to predefined conditions ( age , sex , diabetes mellitus , hypertension , hyperlipidemia , cad , chf , and atrial fibrillation ) , no statistically significant difference between the 2 cohorts with regard to these variables was observed . baseline characteristics between renal stone group and nonrenal stone group in 1998 to 2010 the mean ages of the rs and non - rs cohorts were 47.714.9 and 48.1 14.1 years , respectively . the 2 cohorts showed male predominance ( 65.9% in rs cohort ; 65.8% in non - rs cohort ) . the overall incidence of stroke was higher in the rs cohort compared with the non - rs cohort ( 6.64 vs 6.27 per 1000 person - years , table 2 ) . the relative stroke risk for the rs cohort was 1.06-fold higher than that for the non - rs cohort ( 95% ci = 1.011.11 ) . because the incidence of stroke varied with age and sex , we examined the effects of these variables on stroke in the 2 cohorts . the sex - specific analysis showed that women with rs had a higher stroke incidence than women without rss ( irr = 1.12 , 95% ci = 1.031.21 ) . ci = 1.051.23 ) more prone to stroke than were women in the non - rs group , after adjustment for confounders . the age - specific incidence analysis shows that the rs cohort had a higher hr than the non - rs cohort in the youngest age group did ( 2034 years , hr = 1.47 , 95% ci = 1.101.96 ) , followed by the middle - aged adults group ( 3549 years , hr = 1.12 , 95% ci = 1.001.25 ) . no statistically significant difference of stroke events between the 2 groups was observed for patients aged 50 . hazard ratios and 95% confidence interval of stroke associated with renal stone in cox 's regression analysis we examined the association of nephrolithiasis and stroke type . patients with rss had a significantly higher risk of ischemic stroke ( hr = 1.07 , 95% ci = 1.031.23 ) , particularly women ( hr = 1.13 , 95% ci = 1.031.23 ) , and the younger age group ( 2034 years , hr = 1.45 , 95% ci = 1.012.10 ; 3549 years , hr = 1.19 , 95% ci = 1.041.35 , table 3 ) . hazard ratios and 95% confidence interval of stroke associated with renal stone in cox 's regression analysis in addition , we evaluated whether the risk of stroke increased with the severity of rss . the rss severity was stratified using the data on medical visits for rss and surgical procedures for stone removal . for surgical procedures , compared with the control group , a severity - dependent stroke risk was noted with rs severity stratifications : 1 to 2 surgical procedures , ahr = 0.94 , 95% ci = 0.881.01 ; 34 medical visits , ahr = 8.29 , 95% ci = 6.5010.6 ; > 4 surgical procedures , ahr = 42.5 , 95% ci = 33.853.4 ; p for trend < 0.0001 ( table 4 ) . the risk of stroke among stone patients frequency for medical visits of stones removal in cox proportional hazard regression further , in our study population , we analyzed incidences of deep venous thromboembolism and orthopedic surgeries of lower limbs , which were also risk factors of stroke . among 2343 stroke patients in the rs cohort , there were 25 patients ( 1.07% ) who had deep venous thromboembolism , whereas among 8730 stroke patients in the non - rs cohort , 67 patients ( 0.77% ) had deep venous thromboembolism ( p = 0.16 ) . among 2343 stroke patients in the renal stone cohort , 44 patients ( 1.88 % ) had received orthopedic surgery of lower limbs , whereas among 8730 stroke patients , 174 patients ( 1.99 % ) had received orthopedic surgery of lower limbs ( p = 0.72 ) . a kaplan meier analysis showed that , during the 13-year study period , the overall stroke rate was 54.2% higher for patients with rss who have undergone > 4 surgical procedures for stone removal than for those who did not undergo surgery ( p < 0.0001 , figure 1 ) . previous studies have shown an association between nephrolithiasis and systemic diseases , such as chd , hypertension , and diabetes . the present population - based study enrolling a large number of patients with nephrolithiasis and matched controls from the nhird revealed that nephrolithiasis is associated with an increased risk of ischemic stroke . the patients with rss were predominantly men , consistent with previous studies , indicating that the nhird rs patient population is valid and representative . despite the male predominance in the rs cohort , women with nephrolithiasis had a significantly increased stroke risk . appelros et al reported that men have a higher stroke risk than women . because of the adjustment for the major comorbidities that increase stroke risk in the enrolled population , nephrolithiasis was a significant risk factor of stroke , particularly in women . the sex - specific difference that women with nephrolithiasis are prone to stroke is challenging to explain . however , several studies have reported the sex - specific difference that women with nephrolithiasis have an increased risk of adverse renal outcomes , cardiovascular disease , and hypertension . these observations , along with those of the present study emphasize the strong association of nephrolithiasis with vascular diseases and additional studies to understand the mechanism and validate the effects of nephrolithiasis on associated vascular diseases are warranted . several explanations account for the positive association of nephrolithiasis with stroke in patients > 50 years . first , elderly patients have a high likelihood of being bedridden , immobile , or functionally weak because of traumatic fracture , dementia , or medical illness . second , elderly patients may have a high competing risk between death and stroke than do patients < 50 years . however , the high risk of stroke in the relatively low cardiovascular risk young population with nephrolithiasis emphasizes that the causality of nephrolithiasis is stroke development . this is the first study that determined the association between the severities of nephrolithiasis and stroke . patients who received > 4 surgeries had up to 42.5-fold risk of stroke compared with those treated through conservative medication . thus , stroke risks are significantly apparent in patients with symptomatic nephrolithiasis , emphasizing that nephrolithiasis is linked causally to stroke development . first , the patients with a history of stroke were excluded because a previous stroke is a major predictor for recurrent stroke . extensive matching with 8 confounding variables was defined in selecting the controls to ascertain the association of nephrolithiasis with stroke . second , the follow - up period of 13 years enabled comprehensive evaluation of the time - dependent effects of nephrolithiasis on stroke development . third , an nhi monitoring and auditing system was implemented to supervise insurance claims to prevent over diagnosis and medical resource wastage . based on the accurate diagnosis of the disease of interest and confounding variables , the association between nephrolithiasis and stroke was clearly demonstrated . the limitations of our study include the lack of definite patient information on blood pressure , levels of serum glucose , cad severity , and serum lipids levels . moreover , nhird does not provide information on the lifestyle and personal health behaviors , including smoking , drinking , exercise habits , and obesity ; these variables are known to be related to stroke . however , because nhi covers > 99% of taiwan 's residents , this study generalizes the trend that nephrolithiasis is associated with stroke . in conclusion , a history of renal stones is associated with a significantly increased stroke risk , particularly in women and young patients . the 42.5-fold higher risk of stroke in patients with symptomatic nephrolithiasis requires surgical treatment . the findings can prompt clinical awareness and suggests lifestyle modification to facilitate early prevention of stroke in patients with nephrolithiasis , particularly in women , young population , and those with symptomatic nephrolithiasis patients .

abstractnephrolithiasis is highly prevalent and has been associated with vascular diseases such as cardiovascular events . few studies have comprehensively associated renal stones with stroke.this study explored whether patients with renal stones were at a higher stroke risk than those without renal stones . a national insurance claim dataset of 22 million enrollees in taiwan was used to identify 53,659 patients with renal stones , and 214,107 were selected as age- , sex- , and comorbidity - matched controls for a 13-year follow-up.the relative stroke risk for the rs cohort was 1.06-fold higher than that for the non - rs group ( 95% confidence interval [ ci ] = 1.011.11 ) . age - specific analysis revealed that the adjusted stroke risk for the rs cohort increased as age decreased , with the highest risk of 1.47-fold ( 95% ci = 1.101.96 ) in patients aged 20 to 34 years , followed by a 1.12-fold risk ( 95% ci = 1.001.25 ) in patients aged 35 to 50 years . sex - specific analysis clarified that women in the rs group had a 1.12-fold stroke risk compared with women in the non - rs group ( 95% ci = 1.031.21 ) . patients who had undergone > 4 surgeries had up to 42.5-fold higher risk of stroke ( 95% ci = 33.853.4).the study utilized the national database and demonstrated that patients , particularly women and the younger population , with nephrolithiasis have an increased risk of ischemic stroke development . patients treated with medication or through surgery for rss showed steady and higher risks of stroke than those without surgical or medical intervention . epidemiologic studies of the link between particulate matter ( pm ) concentrations and mortality rates have yielded a range of estimates , leading to disagreement about the magnitude of the relationship and the strength of the causal connection . previous meta - analyses of this literature have provided pooled effect estimates , but have not addressed between - study variability that may be associated with analytical models , pollution patterns , and exposed populations . to determine whether study - specific factors can explain some of the variability in the time - series studies on mortality from particulate matter [ less than / equal to ] 10 microm in aerodynamic diameter ( pm(10 ) ) , we applied an empirical bayes meta - analysis . we estimate that mortality rates increase on average by 0.7% per 10 microg / m(3 ) increase in pm(10 ) concentrations , with greater effects at sites with higher ratios of particulate matter [ less than / equal to ] 2.5 microm in aerodynamic diameter ( pm(2.5))/pm(10 ) . this finding did not change with the inclusion of a number of potential confounders and effect modifiers , although there is some evidence that pm effects are influenced by climate , housing characteristics , demographics , and the presence of sulfur dioxide and ozone . although further analysis would be needed to determine which factors causally influence the relationship between pm(10 ) and mortality , these findings can help guide future epidemiologic investigations and policy decisions.imagesfigure 1

chronic hepatitis b virus ( hbv ) infection is a significant public health threat . approximately 3 - 4 billion people worldwide have been infected with hbv . the natural course of chronic hbv infection is variable , ranging from an inactive hbsag carrier state to a progressive chronic hepatitis which may eventually lead to cirrhosis and hepatocellular carcinoma ( hcc ) . previous research has shown that patients with persistent hbeag - positive state and high dna viral load ( > 10 copy / ml ) are at higher risk of hcc than the hbeag negative individuals with low dna viral load [ 1 , 2 ] . in order to have a better understanding of this phenomenon , we obtained hbsag - positive serum samples from clinical laboratory center of the first people 's hospital of yunnan province , kunming , yunnan , china , from february to august , 2005 . we also followed up a chronically hbv - infected patient who was hbeag positive with high hbv dna load ( 10 copy / ml ) for almost 3 years and studied the evolution of her hbv strains . by sequencing and phylogenetic analysis , we found mutations occurred within the genomes of hbv through time - course evolution . combined with clinical data , whether and how these hbv variants might contribute to viral persistence of infection or liver pathogenesis still needs to be further studied a 31-year - old female with chronic hbv infection was followed up at the first people 's hospital of yunnan province , kunming , yunnan , china . the subject was admitted in the hospital because of allergic purpura in may , 2005 and was diagnosed with chronic infection based on the fact of having been hbsag positive for at least 7 years . her serum samples were collected at three time points after admission : 602 ( 1 day ) , 6022 ( 6 months ) , and 6023 ( two years and seven moths ) and stored at 80c until time of analysis . serological markers of hbv , alt , ast , bil , alb , and glo were analyzed at each time point . the patient was seronegative for antibodies to hav , hcv , hev , and hiv and did not have a history of alcohol abuse , drug abuse , or hepatotoxin exposure . hbv dna was extracted from serum by proteinase k digestion , followed by phenol / chloroform extraction . viral dna load was determined using hbv dna pcr - fluorescence quantitation kit ( kehua bio - tech . the primers used were forward primer : 5ctttttcacctctgcctaatca3 ; reverse primer : 5agaggtgaaaaagttgcatggt3. amplification was performed in a 96-well cycler ( bio - rad , usa ) , and 25 l pcr mixture containing 2.5 mm mgcl2 , 200 m dntp , 400 nm of each primer , and 2 u of ex taq polymerase ( takara bio - tech . the pcr reaction was performed using the following cycles : 94c predenature for 5 min , 30 cycles of 94c for 1 min , 56c for 1 min , 72c for 2 min ; 72c for 10 min as a final extension step . then full - length amplicons were purified using a gel extraction kit ( huashun bio - engineering co. ltd . , the pcr products were purified and cloned in vector pmd18-t ( takara bio - tech . white colonies were picked , correct recombinants were confirmed by pcr , and the double - restriction endonuclease digestion with ecori and hind iii . dna sequencing analysis of the correct recombinants was performed with bigdye terminator v3.1 and 3130 genetic analyzer ( applied biosystems ) ( 7 pairs of sequencing primers can be obtained on request ) . for each time point , 8 - 9 recombinants were randomly selected and sequenced . fourteen hbv complete genomes available in genbank ( genotypes a , b and e to h ) were applied to phylogenetic analysis . additionally , 50 published sequences of genotype c were also used in this study for alignment analysis ( genbank accession numbers can be obtained upon request ) . the whole genome sequences were assembled by bioedit sequence alignment editor software , version 7.0.5.2 . phylogenetic and molecular evolutionary analysis of nucleotide differences within and between the isolate sequences were carried out by mega program , version 3.1 . bootstrap re - sampling and reconstruction were carried out for 1,000 times to confirm the reliability of phylogenetic trees . as showed in table 1 , hbsag and hbeag were positive at all three time points and titers changed during the course of analysis . alt levels showed a tendency of elevation , and at the 3rd time point the level was more than 2 times of the upper limit of normal . while no quantification could be obtained from the 1st time point due to the lack of serum , viral dna load was found to decrease from 10 to 10 copy / ml at the last two time points . 26 sequences were analyzed ( genbank accession number : dq377159 - 377165 , eu306713 - 306714 , eu306722306729 , eu439005 , eu439008439014 and eu439025 ) . the divergence of these 26 sequences was found to be 01.4% . when compared to the 64 complete hbv genome sequences published in the genbank , all 26 sequences were determined to be genotype c and c2 subgroup with a bootstrap value of 100% ( data not shown ) . when compared with the 50 known and complete sequences of genotype c , we found that there were 13 specific nucleotides in all sequenced clones of this patient ( data not shown ) . further analysis of the nucleotide sequence at all three time points indicated that nucleotides t361a , c934a , c2351t / a2353 t , and c2444 t were the mutation been kept in for at least two years ' evolution . these mutations caused nonsense mutation of pres1 and precore / core gene ( t361a and c2444 t , resp . ) , missense mutation of precore / core and pol gene ( c2351t / a2353 t and c934a , resp . ) . nucleotides c339 t and t770c were the new and major mutation at the 3rd time point , and these mutations caused hbsag p62l and polymerase ( pol ) v560a mutation , respectively ( figure 1 ) . premature stop codons of s gene ( g.200c > t , g.565t > a , g.361t > a , g.455c > t , and g.304_305cc > tt ) increased during the course(1/9 versus 3/9 versus 6/8 ) ( figure 2 ) . a1762t / g1764a double mutation existed in all clones , and g1896a existed in 4 clones obtained from all time points . performing alignment showed that over the 2-year period , deletions across overlapping region of polymerase / pres1 ( g.2447_489del 1256 ) or pres1/pres2 ( g.3018_3202del 183 , and g.3144_56del 126 ) , and pre - s1 initiation codon mutations ( g.2846_2865del 18 ) with or without internal deletions mutations ( g.3024_3193del 168 ) were prevalent ( 22/26 , in 85% of the clones ) ( figure 2 ) . all the defective genomes shared sequences from nucleotide position 489 to position 2447 , and common to all genomes is the presence of the complete x gene and the complete c orf . four paires of donor / acceptor were found in the spliced genomes , besides the frequently reported donor sites 2447 and 3018 and the acceptor sites 489 , two new 5-splice donors ( nt3144 and 3024 ) and three new 3-splice acceptors ( nt56 , 3193 and 3202 ) were detected at the 3rd time . up until now , details of the mechanism accounted for the hbv evolution remained unknown . in this study , we followed up a chronically hbv - infected patient and discovered that complex and diverse quasispecies existed in this patient for over two - year disease evolution . based on the alignment and phylogenetic analyses , the divergence and the 13 specific nucleotides of the 26 clones , as well as the phylogenic analysis thus , these results reflect the real evolution process of homogeneous hbv strains in this patient . in the process of evolution , some mutations in this patient were kept in for at least 2 years , and other new mutations emerged at the 3rd time point . these mutations resulted in amino acids substitutions and some caused premature stop codon of the coding regions . among these mutants , the t361a nonsense mutation in the small s region , which resulted in a 69aa truncated hbsag lacking the entire a determinant region ( aa124147 ) , was first detected at the 2nd time point in one clone . at the 3rd time point , however , half of the sequenced clones were found to have this mutation . this mutant has been reported in the occult hbv - infected patients with seronegative for hbsag , and in one case hbv dna was absent from the serum after the two - year [ 7 , 8 ] . in our case , after two - year followup , the hbsag was still at a high level and could be detected by routine immunoassay - based diagnosis . in addition , hbv dna was at a high load despite a slightly decreased level ( 2.4 10 versus 2.5 10 copy / ml ) , and the alt level showed an elevating trend over the study period . in the meanwhile , new point mutations such as c339 t and t770c were detected at the 3rd time point ; the former occurred in the hydrophilic region of hbsag and the latter was previously discovered in a hcc patient ( genbank number ay206393 ) . we speculated that these mutants might afford hbv variants a distinct survival advantage by allowing the mutant virus to escape from the immune system . in addition , these mutations could further transactivate certain oncogenes which in turn rendered the acceleration of the liver damage [ 913 ] . in this patient the deletion region is in the span of hepatocellular - binding site ( aa520 ) and led to aa111 deletion in pres1 . this deletion mutant was previously reported in different clinical cases and genotypes [ 1417 ] . for example , this 18 bp deletion was detected in a heart transplant patient while it was not found with the donor . in our case , retrospective study showed that the son of this patient , though infected with hbv via mother - to - infant transmission , carried none of these mutant strains in the sequenced clones . this phenomenon may imply that host immune pressure was the primary cause of aa111 deletion in pres1 . in addition , this mutant seemed to have reduced from the 1st time point to the 3rd time point ( 7/9 versus 7/9 versus 2/8 ) and had a tendency to be substituted by large fragments deletion located in the pres region at the 3rd time point ( figure 2 ) . whether it was a precursor of large fragment deletion mutation or just an isolated event happened under the immune pressure still needed to be carefully studied . the other prominent variation at the 3rd time point was the defective genomes caused by the large fragment deletion focused on the overlapping regions of polymerase / pres1 and pres1/pres2 . up until now , there have been at least 15 types of spliced hbv dna reported in the literature ( figure 3 ) [ 20 , 21 ] , which consisted of seven 5-splice donors and six 3-splice accepters . in our case , four types of spliced hbv dna were found at the 3rd time point , the donor site and the receptor site of these splicers followed the gt - at rules . among them , the 2447/489 donor / acceptor splicer existed as described in previous reports and produced a presumptive hbv spliced protein ( hbsp ) . in addition , positions 3144 and 3024 as the 5-donor sites and positions 3202 , 3193 , and 56 as the 3-acceptor sites were first reported in our study . during a subsequence screening , we also detected defective genomes in patients with hepatocirrhosis and chb ( genbank accesson numbers : eu439017 , eu306668 , eu306669 , eu330986 , eu306676 , eu306670 ) . though these defective genomes were different in structure , they all shared sequences from nucleotides position 489 to position 2447 . this region contains all known signals required for conventional replication ( dr1 and dr2 ) , synthesis , and processing of pregenomic / c - mrna , prec mrna , and x mrna , and pregenomic rna encapsidation . in contrast , all lacked the complete pre - s1 and , or pre - s2 orfs . many surveys noted that spliced hbv dna and pre - s deletion were more frequently detected in patients with severe liver disease [ 2426 ] and often existed during the process of anti - hbe seroconversion and subsequent loss of hbsag [ 2729 ] . however , in this case with our subsequent screening , all the patients with defective genomes were hbeag positive and with high hbv dna load ( 10 copy / ml ) . based on these findings , we deduced that these defective genomes ( with intact x gene and all signals required for viral life cycle ) might contribute to persistent hbv replication in the patient . other mutations such as c934a , c2351t / a2353 t , c2444 t , a1762t / g1764a , and g1896a were scattered in the whole genomes . in the present study , all clones of the mother from three time points had double mutations of nucleotide a1762t / g1764a in basal core promoter ( bcp ) , four of which were also coupled with g1896a . however , we did not observe any hbeag / anti - hbeag seroconversion at the three time points , nor did we detect the impact of bcp mutation on hbv dna load [ 30 , 31 ] . in addition , multisequences alignment has showed that genotype c had a tendency for higher prevalence of the bcp mutation ( 41 versus 35 , 53.9% ) ( genbank accesson number can be obtained on request ) . combined with the clinic results ( table 1 ) , whether the bcp and precore mutations in this patient were related to infection with genotype c , or whether the patient was seroconverting to anti - hbeag or may develop severe disease [ 32 , 33 ] are questions that still need to be followedup . in conclusion , our data showed that viral quasispecies are very closely related to genomes but exist in an environment of mutation , selection , and competition , and thus lead to a dynamic and changing population . during this progress , some mutations were kept in and became the dominant strains , some new mutations emerged , and these strains may play important roles in the persistence of infection or the progression of disease . hepatocellular carcinoma ( hcc ) is one of the most lethal malignancies , and surgical resection improves the survival rates for patients . however , the prognosis after surgical resection of hcc remains poor because of high recurrence rates . using biomarkers to identify patients presenting with a higher risk of poor prognosis may reduce mortality after curative hepatectomy . although there are many reports on histologic parameters for predicting hcc prognosis , molecular markers for hcc recurrence and prognosis could provide additional information . cancer cells frequently express genes that are specifically or preferentially expressed in male germ cells under normal conditions . atpase family aaa domain - containing 2 ( atad2 ) is one such , and works as an important cofactor for myc - dependent transcription . through myc and e2f transcription factors , atad2 increases the expression of proliferation - related and anti - apoptotic genes in many different types of cancer , including breast carcinoma , non - small cell lung carcinoma , and prostate carcinoma [ 5 - 8 ] . , atad2 has been identified as a candidate driver gene located within the amplified 8q24 locus . huang et al . reported that a novel highly up - regulated exon - exon junction was detected in atad2 gene by rna - seq and the gene was highly expressed in hcc tissues . a recent study found that the high expression of atad2 in hcc was an independent predictor of shortened overall survival . however , the prognostic significance of atad2 protein expression in hcc is unclear . in the present study , we evaluated atad2 protein expression by immunohistochemistry to elucidate the prognostic role of atad2 in 182 hcc patients with long - term follow - up . hcc tissue samples were collected from 182 patients who were treated with surgical resection between july 2000 and may 2006 at samsung medical center ( seoul , korea ) . inclusion criteria were histologically confirmed hcc and curative resection of tumor without preoperative or postoperative adjuvant therapy . we defined curative resection as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors as indicated by a computed tomography scan one month after surgery . microvascular invasion was considered present when at least one or more endothelial cells or the tunica media of the vessel surrounded a neoplastic cell group . tumor stage was determined by both the american joint committee on cancer ( ajcc ) staging system and barcelona clinic liver cancer ( bclc ) staging classification . using 2 years as the cut off , all hcc patients were followed by monitoring serum -fetoprotein levels and three phase dynamic computed tomography scans or magnetic resonance imaging every three months after surgery . the follow - up period for recurrence was at least 18 months , and the median follow - up period was 120.0 months ( range , 14.0 to 151.4 months ) for survivors . recurrence - free survival ( rfs ) was defined as from the date of resection until the detection of tumor recurrence . we chose disease - specific death ( hcc - related death ) as the clinical endpoint for survival analysis , defined as : ( 1 ) tumor occupying more than 80% of the liver , ( 2 ) portal venous tumor thrombus proximal to the second bifurcation , ( 3 ) obstructive jaundice due to the tumor , ( 4 ) distant metastases , or ( 5 ) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation . disease - specific survival ( dss ) was defined from the date of resection to the date of hcc - related death . histologic sections were examined by two pathologists and representative tumor regions were marked in the formalin - fixed paraffin - embedded blocks . two tissue cores measuring 2.0 mm in diameter were punched from the marked areas of each block and arranged into new paraffin blocks . as controls , two cores of normal liver tissue from 12 patients with metastatic colon carcinoma of the liver were included in each array block . antigen retrieval was performed with 0.01 mol / l citrate buffer at ph 6.0 for 30 minutes in a pressure cooker . sections were incubated with a rabbit polyclonal antibody to atad2 ( 1:100 , nbp1 - 84122 , novus biologicals , littleton , co ) for 60 minutes at room temperature . the positive control ( human normal testis ) showed intense nuclear atad2 expression in spermatogenic cells of seminiferous tubules . no immunoreactivity was observed in tissue sections used as negative control where the primary antibody was replaced by preimmune rabbit serum . in order to validate the concordance between tissue microarrays and whole tumor sections , we observed atad2 expression for 40 corresponding whole tumor sections randomly chosen from the 182 cases . and h.w.h . ) who were blinded to the clinical details , and any discrepancies were resolved by consensus . a nearly homogeneous nuclear immunostaining with moderate staining intensity was observed . for determining atad2 expression , the proportion of stained tumor cells was determined semi - quantitatively and each sample was scored on a scale of 0 - 4 ( 0 , < 5% ; 1 , 5%-25% ; 2 , 26%-50% ; 3 , 51%-75% ; 4 , > 75% ) . duplicate tissue cores for each tumor showed high levels of homogeneity for proportion of stained cells . the association of atad2 expression with clinicopathologic features was examined by the chi - square test or fisher exact test . univariate and multivariate analyses of risks for tumor recurrence were performed using the logistic regression model . cumulative survival rates were calculated by the kaplan - meier method and analyzed by the log - rank test . the cox proportional hazards regression model was used to assess the association of survival time regressed upon multiple clinicopathologic variables . variables that were statistically significant in the univariate analysis were included in the multivariate analysis . hcc tissue samples were collected from 182 patients who were treated with surgical resection between july 2000 and may 2006 at samsung medical center ( seoul , korea ) . inclusion criteria were histologically confirmed hcc and curative resection of tumor without preoperative or postoperative adjuvant therapy . we defined curative resection as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors as indicated by a computed tomography scan one month after surgery . microvascular invasion was considered present when at least one or more endothelial cells or the tunica media of the vessel surrounded a neoplastic cell group . tumor stage was determined by both the american joint committee on cancer ( ajcc ) staging system and barcelona clinic liver cancer ( bclc ) staging classification . using 2 years as the cut off , all hcc patients were followed by monitoring serum -fetoprotein levels and three phase dynamic computed tomography scans or magnetic resonance imaging every three months after surgery . the follow - up period for recurrence was at least 18 months , and the median follow - up period was 120.0 months ( range , 14.0 to 151.4 months ) for survivors . recurrence - free survival ( rfs ) was defined as from the date of resection until the detection of tumor recurrence . we chose disease - specific death ( hcc - related death ) as the clinical endpoint for survival analysis , defined as : ( 1 ) tumor occupying more than 80% of the liver , ( 2 ) portal venous tumor thrombus proximal to the second bifurcation , ( 3 ) obstructive jaundice due to the tumor , ( 4 ) distant metastases , or ( 5 ) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation . disease - specific survival ( dss ) was defined from the date of resection to the date of hcc - related death . histologic sections were examined by two pathologists and representative tumor regions were marked in the formalin - fixed paraffin - embedded blocks . two tissue cores measuring 2.0 mm in diameter were punched from the marked areas of each block and arranged into new paraffin blocks . as controls , two cores of normal liver tissue from 12 patients with metastatic colon carcinoma of the liver were included in each array block . antigen retrieval was performed with 0.01 mol / l citrate buffer at ph 6.0 for 30 minutes in a pressure cooker . sections were incubated with a rabbit polyclonal antibody to atad2 ( 1:100 , nbp1 - 84122 , novus biologicals , littleton , co ) for 60 minutes at room temperature . the positive control ( human normal testis ) showed intense nuclear atad2 expression in spermatogenic cells of seminiferous tubules . no immunoreactivity was observed in tissue sections used as negative control where the primary antibody was replaced by preimmune rabbit serum . in order to validate the concordance between tissue microarrays and whole tumor sections , we observed atad2 expression for 40 corresponding whole tumor sections randomly chosen from the 182 cases . all sections were scored by two independent pathologists ( c .- k.p . and h.w.h . ) who were blinded to the clinical details , and any discrepancies were resolved by consensus . a nearly homogeneous nuclear immunostaining with moderate staining intensity was observed . for determining atad2 expression , the proportion of stained tumor cells was determined semi - quantitatively and each sample was scored on a scale of 0 - 4 ( 0 , < 5% ; 1 , 5%-25% ; 2 , 26%-50% ; 3 , 51%-75% ; 4 , > 75% ) . duplicate tissue cores for each tumor showed high levels of homogeneity for proportion of stained cells . when there were differences between duplicate tissue cores the association of atad2 expression with clinicopathologic features was examined by the chi - square test or fisher exact test . univariate and multivariate analyses of risks for tumor recurrence were performed using the logistic regression model . cumulative survival rates were calculated by the kaplan - meier method and analyzed by the log - rank test . the cox proportional hazards regression model was used to assess the association of survival time regressed upon multiple clinicopathologic variables . variables that were statistically significant in the univariate analysis were included in the multivariate analysis . the mean age of the 182 hcc patents was 52.4 years ( range , 17 to 76 years ) , and 82.4% of the patients were male . chronic hepatitis b virus infection was detected in 141 patients ( 77.5% ) and chronic hepatitis c virus infection in 16 patients ( 8.8% ) . the mean tumor size was 5.3 cm , and 109 of the 182 tumors ( 59.9% ) were 5 cm in size . tumor recurrence was detected in 132 patients ( 72.5% ) , early recurrence in 108 patients ( 59.3% ) , and late recurrence in 24 patients ( 13.2% ) . fifteen of the 91 deaths in this study were due to non - hcc causes . nine of the 15 deaths were due to hepatic failure , five due to non - hepatic causes , and one due to unknown cause . immunoreactivity for atad2 was observed only in the nuclei of tumor cells with moderate staining intensity . we regarded the atad2 as positive when 5% of tumor cells showed nuclear immunoreactivity ( fig . atad2 expression was significantly associated with larger tumor size ( p=0.001 ) , higher edmondson grade ( p=0.015 ) , microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p atad2 expression was associated with the early recurrence ( p < 0.001 ) , but not with the late recurrence ( p=0.619 ) ( table 1 ) . in univariate analyses , early recurrence was significantly associated with larger tumor size ( p=0.019 ) , microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p < 0.001 ) , higher ajcc t - stage ( p < 0.001 ) , lower albumin level ( p=0.041 ) , viral etiology ( p=0.004 ) , and atad2 expression ( p < 0.001 ) . as ajcc t - stage and bclc stage were associated with vascular invasion , we did not make multiple analyses with these variables to avoid potential bias . an evaluation of the association of serum -fetoprotein level with early recurrence was not performed due to missing data ( n=175 ) . in multivariate analyses , microvascular invasion ( p=0.003 ) , intrahepatic metastasis ( p=0.003 ) , and viral etiology ( p=0.029 ) were independent predictors of early recurrence . atad2 expression tended to be independent predictor of early recurrence ( p=0.059 ) ( table 2 ) . the rfs and dss rates for 182 hcc patients were 36.1% and 75.5% at 3 years , 32.1% and 66.7% at 5 years , 27.5% and 61.1% at 7 years , and 26.6% and 55.2% at 9 years , respectively . on univariate analyses , larger tumor size , microvascular invasion , major portal vein invasion , intrahepatic metastasis , higher ajcc t - stage , higher bclc stage , and lower albumin level showed unfavorable influences on both rfs and dss . the 5-year rfs rate of the atad2-positive group was significantly lower than that of the atad2-negative group ( 23.1% vs. 48.6% ) ( fig . the mean rfs of atad2-positive group was 37.6 months and the atad2-negative group was 74.0 months . subgroup analysis among patients with tumor size 5.0 cm ( n=109 ) , patients at bclc stage 0 or a ( n=92 ) , and patients with -fetoprotein 20 ng / ml ( n=61 ) , the atad2-positive groups ( n=61 , n=50 , and n=30 , respectively ) also unfavorably influenced rfs ( p=0.008 , p=0.009 , and p=0.013 , respectively ) ( fig . the 5-year dss rate of the atad2-positive group was significantly lower than that of the atad2-negative group ( 57.8% vs. 83.4% ) ( fig . the mean dss of atad2-positive group was 88.1 months and of atad2-negative group , 123.3 months . multivariate analyses indicated that intrahepatic metastasis and lower albumin level were found to be independent predictors of both shorter rfs and shorter dss . atad2 expression ( p=0.002 ) was an independent predictor of shorter rfs , but not of dss ( p=0.109 ) . atad2-positive patients were more likely to suffer from recurrence than atad2-negative patients ( hazard ratio , 1.857 ) ( table 4 ) . the mean age of the 182 hcc patents was 52.4 years ( range , 17 to 76 years ) , and 82.4% of the patients were male . chronic hepatitis b virus infection was detected in 141 patients ( 77.5% ) and chronic hepatitis c virus infection in 16 patients ( 8.8% ) . the mean tumor size was 5.3 cm , and 109 of the 182 tumors ( 59.9% ) were 5 cm in size . tumor recurrence was detected in 132 patients ( 72.5% ) , early recurrence in 108 patients ( 59.3% ) , and late recurrence in 24 patients ( 13.2% ) . fifteen of the 91 deaths in this study were due to non - hcc causes . nine of the 15 deaths were due to hepatic failure , five due to non - hepatic causes , and one due to unknown cause . immunoreactivity for atad2 was observed only in the nuclei of tumor cells with moderate staining intensity . we regarded the atad2 as positive when 5% of tumor cells showed nuclear immunoreactivity ( fig . atad2 expression was significantly associated with larger tumor size ( p=0.001 ) , higher edmondson grade ( p=0.015 ) , microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p < 0.001 ) , higher ajcc t - stage ( p atad2 expression was associated with the early recurrence ( p < 0.001 ) , but not with the late recurrence ( p=0.619 ) ( table 1 ) . in univariate analyses , early recurrence was significantly associated with larger tumor size ( p=0.019 ) , microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p < 0.001 ) , higher ajcc t - stage ( p < 0.001 ) , lower albumin level ( p=0.041 ) , viral etiology ( p=0.004 ) , and atad2 expression ( p < 0.001 ) . as ajcc t - stage and bclc stage were associated with vascular invasion , we did not make multiple analyses with these variables to avoid potential bias . an evaluation of the association of serum -fetoprotein level with early recurrence was not performed due to missing data ( n=175 ) . in multivariate analyses , microvascular invasion ( p=0.003 ) , intrahepatic metastasis ( p=0.003 ) , and viral etiology ( p=0.029 ) were independent predictors of early recurrence . atad2 expression tended to be independent predictor of early recurrence ( p=0.059 ) ( table 2 ) . the rfs and dss rates for 182 hcc patients were 36.1% and 75.5% at 3 years , 32.1% and 66.7% at 5 years , 27.5% and 61.1% at 7 years , and 26.6% and 55.2% at 9 years , respectively . on univariate analyses , larger tumor size , microvascular invasion , major portal vein invasion , intrahepatic metastasis , higher ajcc t - stage , higher bclc stage , and lower albumin level showed unfavorable influences on both rfs and dss . the 5-year rfs rate of the atad2-positive group was significantly lower than that of the atad2-negative group ( 23.1% vs. 48.6% ) ( fig . the mean rfs of atad2-positive group was 37.6 months and the atad2-negative group was 74.0 months . subgroup analysis among patients with tumor size 5.0 cm ( n=109 ) , patients at bclc stage 0 or a ( n=92 ) , and patients with -fetoprotein 20 ng / ml ( n=61 ) , the atad2-positive groups ( n=61 , n=50 , and n=30 , respectively ) also unfavorably influenced rfs ( p=0.008 , p=0.009 , and p=0.013 , respectively ) ( fig . the 5-year dss rate of the atad2-positive group was significantly lower than that of the atad2-negative group ( 57.8% vs. 83.4% ) ( fig . the mean dss of atad2-positive group was 88.1 months and of atad2-negative group , 123.3 months . multivariate analyses indicated that intrahepatic metastasis and lower albumin level were found to be independent predictors of both shorter rfs and shorter dss . atad2 expression ( p=0.002 ) was an independent predictor of shorter rfs , but not of dss ( p=0.109 ) . atad2-positive patients were more likely to suffer from recurrence than atad2-negative patients ( hazard ratio , 1.857 ) ( table 4 ) . atad2 contributes to the development of aggressive cancer through the enhancement of myc - dependent transcription . atad2 expression is high in several human cancers , including breast carcinoma , colon carcinoma , lung carcinoma , stomach carcinoma , and uterine carcinoma . high atad2 expression is a strong predictor of rapid mortality in lung and breast cancers . atad2 depletion by rna interference knockdown reduced huh7 and hcclm3 cell proliferation and led to a g1 phase cell cycle arrest . in hcc cells , atad2 regulates cell migration / invasion via the regulation of apc and/or ctnna1 expression . in this study , we evaluated the prognostic significance of atad2 protein expression in hcc and demonstrated that atad2 expression is significantly associated with larger tumor size , higher edmondson grade , microvascular invasion , intrahepatic metastasis , higher ajcc t - stage , and higher bclc stage . moreover , atad2 expression is an independent predictor of shorter rfs and tended to predict early recurrence . in the present study , early stage hcc patients with tumor size 5.0 cm or patients at bclc stage 0 or a have favorable outcomes in both rfs and dss ( table 3 ) , but 75 of 109 ( 68.8% ) with tumor size 5.0 cm and 59 of 92 ( 64.1% ) bclc stage 0 or a developed tumor recurrence . we found within these populations , atad2 positivity unfavorably influenced rfs ( p=0.008 and p=0.009 , respectively ) . identification of patients with poor prognosis in early stage hcc is critical to the optimization of personalized treatment . thus , our findings support the possible importance of atad2 expression in hcc in detecting a phenotype that can predict recurrence risk in early stage hcc . serum -fetoprotein is widely used to screen , mainly for hcc , and is an important predictor of patient survival following tumor resection . preoperative high serum -fetoprotein level ( > 60 ng / ml ) could be a risk factor for recurrence after resection in patients with liver cirrhosis . however , there is no effective marker for monitoring recurrence for patients with normal serum -fetoprotein level ( 20 ng / ml ) after hepatectomy . in this study , tumor recurrence was detected in 40 of 61 patients ( 65.6% ) with normal serum -fetoprotein level . we found that within patients with normal serum -fetoprotein level , the atad2-positive group still had significantly poorer rfs than the atad2-negative group ( p=0.013 ) . thus , our study revealed the potential use of atad2 in predicting recurrence risk in the patient group in which serum -fetoprotein levels were not prognostically predictive . our findings indicate that atad2 protein may be a novel potential predictor of rfs in hcc patients after curative resection , and atad2 may have prognostic value in hcc patients with early stage hcc or normal serum -fetoprotein level . further study is needed to examine the roles of atad2 protein expression in the development and progression of hcc .

mutations in full - length hbv isolates obtained from a chronic hbv - infected patient were evaluated at three time points : 1 day , 6 months , and 31 months . while 5 nucleotides variation , and an 18 bp deletion of pres1 have been kept in during at least the first two years , c339 t mutation occurring in the hydrophilic region of hbsag and t770c that caused polymerase v560a substitution were the new point mutations found existing in sequenced clones of the 3rd time point . internal deletion of coding region obviously appeared in the 3rd time point . the splicers included two new 5-splice donors and three new 3-splice acceptors besides the reported donors and acceptors and may have produced presumptive hbv - spliced proteins or truncated pres proteins . alt , hbeag and viral dna load varied during the follow - up years . these data demonstrated the diversity of genomes in hbv - infected patient during evolution . combined with clinical data , the hbv variants discovered in this patient may contribute to viral persistence of infection or liver pathogenesis . purposecancer cells frequently express genes that are specifically or preferentially expressed in male germ cells under normal conditions . the atpase family aaa domain - containing 2 ( atad2 ) is one such and works as an important cofactor for myc - dependent transcription . in hepatocellular carcinoma ( hcc ) , atad2 has been identified as a candidate driver gene located within the amplified 8q24 locus . however , the prognostic significance of atad2 protein expression in hcc remains uncertain.materials and methodswe investigated atad2 protein expression by immunohistochemistry in tumor tissue from 182 hcc patients who underwent curative resection . associations of atad2 expression with clinicopathologic variables or prognosis of hcc patients were analyzed.resultsatad2 expression was observed in 119 ( 65.4% ) of the 182 hccs and tended to be independent predictor of early recurrence ( p=0.059 ) . atad2 expression showed an unfavorable influence on recurrence - free survival ( rfs ) ( p < 0.001 ) . subgroup analysis among patients with tumor size 5.0 cm ( n=109 ) , patients at barcelona clinic liver cancer stage 0 or a ( n=92 ) , and patients with -fetoprotein 20 ng / ml ( n=61 ) , the atad2-positive groups unfavorably influenced rfs ( p=0.008 , p=0.009 , and p=0.013 , respectively ) . in addition , atad2 expression was an independent predictor of shorter rfs ( p=0.002 ) . atad2 expression showed an unfavorable influence on disease - specific survival ( p=0.001 ) , but was not an independent predictor of shorter disease - specific survival ( p=0.109).conclusionatad2 protein expression may be a potential predictor of rfs in hcc patients after curative resection and atad2 may have prognostic value in patients with early stage hcc or normal serum -fetoprotein level .

subjective tinnitus , a phantom sound , is often described as a ringing , hissing , or buzzing sensation . in the united states , an estimated 50 million adults have experienced tinnitus occasionally and 16 million experience it chronically . patients with chronic tinnitus often suffer from sleep disturbance , depression , and anxiety , conditions that negatively impact the quality of life . since tinnitus often persists even after sectioning the auditory nerve and since tinnitus masking profiles differ from external sounds , aberrant neural activity in the central nervous system ( cns ) rather than the cochlea is believed to play a major role in its development and maintenance [ 68 ] . on the basis of previous electrophysiological and neuroimaging studies [ 9 , 10 ] , tinnitus is believed to be generated by aberrant neural activity in the central auditory pathway by a variety of mechanisms such as increased spontaneous activity , increased neural synchrony , altered tonotopy , aberrant neural connectivity between auditory and nonauditory structures , aberrant attentional , or gating mechanisms . the fact that some patients are constantly aware of their tinnitus and find it extremely disturbing suggests that attentional or emotional neural networks may contribute to the severity of tinnitus distress . since subjective tinnitus is an endogenous ongoing process , resting - state functional magnetic resonance imaging ( fmri ) has proved to be a useful noninvasive technique for determining how structurally segregated and functionally specialized cerebral centers are interconnected as reflected by low - frequency ( 0.010.1 hz ) fluctuations in blood - oxygenation - level dependent ( bold ) signals [ 1315 ] . previous studies have employed resting - state fmri to examine multiple whole - brain networks such as the attention network and default mode network ( dmn ) to study the neural mechanisms of tinnitus . kim et al . found increased functional connectivity between the resting - state attention network and an auditory network suggesting that this network might contribute to the perception or salience of tinnitus . burton et al . proposed that tinnitus associated with hearing loss was the result of usage - induced changes in the organization of sensory networks and interference with the ventral and dorsal attention networks and executive control of attention ( eca ) networks . because of its persistent nature , some have proposed that tinnitus generators become integrated with the dmn [ 18 , 19 ] . also identified tinnitus - specific alterations in the connectivity encompassing the dmn and dorsal attention network . therefore , the above studies suggest that the attention network as well as the dmn may play a pivotal role in the neurological pathophysiology of tinnitus . moreover , our group found that chronic tinnitus patients with normal hearing out to the extended high frequencies exhibited aberrant amplitude of low - frequency fluctuations ( alff ) in specific dmn regions including the middle temporal gyrus ( mtg ) and angular gyrus ; the magnitude of the alff alterations was correlated with tinnitus severity . one limitation of previous tinnitus studies is that they mainly measured the interregional synchronization between distinct brain areas or correlations between resting - state connectivity networks while few have focused on investigating abnormal intraregional activity in specific brain regions . regional homogeneity ( reho ) analysis , a robust algorithm that quantifies the local synchronization within neighboring voxels during resting state , can be used to identify aberrant local neural activity coherence across the whole brain compared with the functional connectivity analyses . altered reho values may relate to disequilibrium of spontaneous neural activity within and between corresponding brain regions . indeed , aberrant reho values , indicative of disrupted local functionality , have been linked to several neurological impairments such as alzheimer 's disease , parkinson 's disease , schizophrenia , and hepatic encephalopathy [ 2326 ] . therefore , reho measurement is a potentially powerful tool to detect aberrant resting - state brain activity which complements information provided by functional connectivity analysis . in this study , we explored the intra- and interregional synchronization of multiple cerebral networks by combining reho and functional connectivity analyses to gain additional insights regarding aberrant neural activity in chronic tinnitus patients with normal hearing thresholds . we speculated that ( 1 ) abnormal reho and functional connectivity would be detected within tinnitus - related networks such as the attention network and dmn and ( 2 ) these abnormalities in the resting - state networks would be correlated with specific tinnitus characteristics such as tinnitus distress . thirty chronic tinnitus patients and 30 healthy subjects without tinnitus were recruited through newspaper advertisements and community health screenings from september 2011 to september 2013 . one of the tinnitus patients was subsequently excluded from the study because of the exceeded limits for head motion during mr scanning . the tinnitus patients and healthy subjects were group - matched with respect to age , sex , and education . twelve patients reported predominantly left - sided tinnitus , 6 reported predominantly right - sided tinnitus , and 11 described their tinnitus as bilateral or originating within the head . the severity of tinnitus and related distress were assessed by the iowa version of the tinnitus handicap questionnaires ( thq ) . all the participants had no hearing loss in any of 10 measured audiometric frequencies ranging from 250 hz to 16 khz ( hearing thresholds < 25 db ) none of the participants had symptoms of depression or anxiety based on the self - rating depression scale ( sds ) and self - rating anxiety scale ( sas ) ( overall scores < 50 ) [ 28 , 29 ] . participants were excluded if they reported suffering from hyperacusis , meniere 's diseases , or pulsatile tinnitus or if they had a past history of heavy smoking , alcoholism , stroke , head injury , parkinson 's disease , alzheimer 's disease , major depression , epilepsy , or other neurological or psychiatric illness , major medical illness ( e.g. , anemia , thyroid dysfunction , and cancer ) , mri contraindications , and severe visual impairment . table 1 summarizes the characteristics of the chronic tinnitus patients and healthy subjects . this study was approved from the research ethics committee of the affiliated zhongda hospital of southeast university . mri data were acquired using a 3.0-tesla trio scanner ( siemens , erlangen , germany ) . the earplugs ( hearos ultimate softness series , usa ) were used to attenuate scanner noise by approximately 32 db . subjects were instructed to keep their eyes closed without falling asleep , not to think of anything in particular , and to avoid any head motion during the scan . structural images were obtained using a t1-weighted 3d spoiled gradient - echo sequence ( repetition time = 1900 ms ; echo time = 2.48 ms ; thickness = 1 mm ; gap = 0 mm ; acquisition matrix = 256 256 ; slices = 176 ; flip angle = 90 ; field of view = 250 mm 250 mm ) . functional images were acquired using a gradient - echo planar sequence ( repetition time = 2000 ms ; echo time = 25 ms ; thickness = 4 mm ; gap = 0 mm ; acquisition matrix = 64 64 ; slices = 36 ; flip angle = 90 ; field of view = 240 mm 240 mm ) . briefly , the unified segmentation model was used to segment cerebral tissues into gray matter ( gm ) , white matter ( wm ) , and cerebrospinal fluid ( csf ) . functional data were preprocessed with the toolbox data processing assistant for resting - state fmri programs through statistical parametric mapping ( spm8 ; http://www.fil.ion.ucl.ac.uk/spm/ ) and resting - state fmri data analysis toolkit ( rest1.8 ; http://www.restfmri.net/ ) . the first 10 volumes were discarded and the remaining 230 consecutive volumes were used for data analysis . afterwards , the procedures were carried out as follows : slice - timing adjustment , realignment for head - motion correction , spatially normalized into the stereotactic space of the montreal neurological institute ( mni ) ( resampling voxel size = 3 3 3 mm ) and smoothed using a gaussian kernel of 4 mm full width at half - maximum ( fwhm ) , detrending , and filtering ( 0.010.08 hz ) . the participants who had a head motion more than 2.0 mm displacement or a 2.0 rotation in the x , y , or z directions were excluded from this study . briefly , kendall 's coefficient of concordance ( kcc ) was computed to measure the local synchronization of a given voxel with 26 nearest neighboring voxels in a voxelwise way , and the reho value was assigned to the central voxel . individual reho maps were divided by the global mean value within the whole brain mask for normalization . then , the normalized reho maps were spatially smoothed with a gaussian kernel of 4 mm fwhm . a two - sample t - test was performed on the group reho maps in a voxel - by - voxel way to explore the reho differences between two groups . a threshold was corrected p < 0.01 , with a monte carlo simulation for multiple comparisons ( http://afni.nimh.nih.gov/pub/dist/doc/manual/alphasim.pdf/ ) . age , sex , education , and gm volume were included as nuisance covariates , to control for the possible influences of these four factors on the results . four areas based on the increased reho findings were defined as regions of interest ( roi ) , including the bilateral anterior insular cortex ( ai ) , the left inferior frontal gyrus ( ifg ) , and the right supramarginal gyrus ( smg ) . then , pearson 's correlation coefficients were computed between the mean signal change of each roi and the time series of each voxel . finally , the correlation coefficients were converted into z values using fisher z - transform to improve the normality . six parameters of head motion and average time courses of global , wm , and csf signals were removed by linear regression analysis . the individual z values were entered into random effect one - sample t - tests to identify the brain regions exhibiting significant connectivity to each roi at a threshold of p < 0.01 ( alphasim correction ) . two - sample t - tests were performed to explore the differences of the functional connectivity of each roi between tinnitus patients and controls . a mask was created for two - sample t - tests by combining the regions exhibiting significant connectivity to each roi . the brain connectivity graphs were visualized using the brainnet viewer ( http://www.nitrc.org/projects/bnv/ ) . between - group t - test and -test were used to compare demographic and clinical data ( p < 0.05 was considered to be significant ) . to investigate the relationship between fmri data and clinical characteristic of tinnitus patients , the regions showing significant increased reho or functional connectivity between tinnitus patients and healthy controls were extracted . mean reho or z values within these clusters were correlated against each clinical characteristic of tinnitus patients using pearson 's correlation analysis by spss software ( version 18.0 ; spss , chicago , il , usa ) . partial correlations were calculated after correction for age , sex , education , and gm volume . table 2 compares the brain volumes of the chronic tinnitus patients and healthy controls . the gm , wm , and brain parenchyma volumes in subjects with tinnitus were not significantly different from healthy controls . compared to the healthy controls , chronic tinnitus patients had significantly increased reho values in the bilateral ai , left ifg , and right smg . in contrast , decreased reho values in tinnitus patients were observed in the left cuneus ( figure 1(a ) and table 3 ) . based on increased reho values in tinnitus patients , we defined four rois for our connectivity analysis : left ai , right ai , left ifg , and right smg . tinnitus patients compared to controls showed significantly greater connectivity between the seed region in left ai and the left mfg , right inferior temporal gyrus ( itg ) , and right precuneus ( figures 1(b ) and 2(c ) and table 4 ) . relative to controls , tinnitus patients showed significantly greater connectivity between the seed region in right ai and the right mfg , right superior temporal gyrus ( stg ) , left precuneus , and left posterior cingulate cortex ( pcc ) . tinnitus patients relative to controls also demonstrated significantly increased connectivity between the seed region in left ifg and the right mfg , right itg , and right anterior cingulate cortex ( acc ) . finally , tinnitus patients compared to controls exhibited significantly greater connectivity between the seed region in the right smg and the left ifg and right orbitofrontal cortex ( ofc ) . in the tinnitus patients , there were no significant correlations between abnormal reho values and any of the tinnitus characteristics . however , the change in functional connectivity between the left ai and the left mfg was positively correlated with the thq score ( r = 0.459 , p = 0.012 ) ( figure 2 ) . in addition , the increased functional connectivity between the right ai and the right mfg was also positively correlated with the thq score ( r = 0.479 , p = 0.009 ) . none of the other regions of increased functional connectivity were significantly correlated with thq scores . moreover , none of the regions of increased functional connectivity were correlated with sds or sas scores or tinnitus duration . this present study investigated for the first time altered intra- and interregional synchronization of several cerebral networks related to tinnitus by combining resting - state reho , a measure of local synchrony , and seed - based functional connectivity analyses . we detected abnormal reho and functional connectivity within specific brain regions belonging to the attention network and the dmn . furthermore , the abnormalities involved in the attention network showed significant correlations with the tinnitus distress . subjective tinnitus is generally associated with hearing loss which decreases the neural output of the cochlea . peripheral damage , in turn , is believed to trigger aberrant neuroplastic changes in the cns that result in tinnitus [ 10 , 11 ] . however , some tinnitus patients have normal hearing out to the extended high frequencies and no obvious signs of hearing pathologies raising questions about the source of the aberrant neural activity involved in their phantom percepts . to address this issue , we obtained structural and functional imaging data from normal controls and tinnitus patients , both with normal hearing thresholds out to the extended high frequencies and no evidence of hyperacusis or other medical problems . thus , the tinnitus patients evaluated in this study may represent a somewhat unique tinnitus phenotype different from others with mild hearing loss or hyperacusis [ 35 , 36 ] . since tinnitus has been associated with structural changes , we compared gm and wm volumes but did not detect any differences between our normal hearing tinnitus patients and matched controls . previous studies have reported decreases in gm volume in tinnitus patients in several brain regions including acc , nucleus accumbens , ventromedial prefrontal cortex , inferior colliculus , hippocampus , superior frontal gyrus , occipital lobe , hypothalamus , and heschl 's gyrus [ 3741 ] . increases in gm volume have also been observed in tinnitus patients in the stg and mtg . however , the changes in gm volume seen in these tinnitus patients were typically correlated with hearing loss particularly when testing was extended out beyond 8 khz [ 37 , 38 , 41 , 42 ] . since we did not detect significant differences in gm volume between our tinnitus patients and controls , the most parsimonious explanation for this is the absence of any hearing loss out to 16 khz and the absence of hyperacusis in our tinnitus patients . however , an alternative possibility is that our analytical techniques were not sensitive enough to detect regional differences in gm volume or intensity in our tinnitus patients [ 35 , 41 ] . regardless of which explanation is correct , our results suggest that abnormal functional neural networks can exist prior to major structural alterations in tinnitus patients with normal hearing . in the present study , the reho is a data - driven method used to measure the extent to which brain activity is synchronized within a cluster of voxels , that is , neural synchronization within a specific brain region . reho analyzes the regional connectivity of the neuronal tissue and may be seen as a measure of the smallest network integrity . altered reho is possibly related to the changes of temporal aspects of the spontaneous neuronal activity in the regional brain . therefore , increased or decreased reho reflects the local destruction of the synchronization of spontaneous neuronal activity in certain regions and implies functional deficits . increased local synchrony , as reflected in reho , could be a critical factor in initiating tinnitus since it might reflect the increased coherence of spontaneous neuronal activity [ 22 , 43 ] . enhanced local synchrony , or increased reho , could develop within a small cluster of neurons because of increased coupling of local factors such as loss of local intracortical inhibition , increased local intracortical excitation , aberrant receptors , and anatomical rewiring or plasticity - induced experience [ 4451 ] . local synchronization in one region of the brain could entrain other areas with preexisting long - range connections and reciprocal feedback circuits could further enhance interregional coupling [ 1 , 5254 ] . in some models , enhanced local synchrony results from inhomogeneities in cortical circuitry induced by cochlear hearing loss . these models would be difficult to reconcile with the absence of hearing loss in our patients . however , certain cochlear pathologies involving damage to the inner hair cells or auditory nerve fibers can go undetected by pta pointing out the need for more sophisticated testing to detect hidden hearing loss [ 55 , 56 ] . in tinnitus patients , reho values were significantly enhanced in four regions , the left and right ai , left ifg , and right smg . the ai and the ifg are key nodes in the attention networks , specifically the eca network . on the basis of resting - state quantitative electroencephalography ( qeeg ) , the ai and ifg have been implicated in tinnitus and specific tinnitus characteristics [ 5759 ] . moreover , tinnitus questionnaire scores were correlated to heart rate variability markers and related to bilateral neural activity in ai . greater synchrony of alpha activity was observed bilaterally in the ai of patients with more severe tinnitus - related distress . ai , which serves in the cingulo - opercular network as well as in the attention network , exhibits altered intra- and interregional synchronization in a number of pathological situations [ 6163 ] . similarly , the vital role of the frontal cortex in subserving tinnitus mechanism has been postulated [ 1 , 45 , 52 ] . previous research has already shown that the ifg is important for emotional processing of sounds [ 64 , 65 ] . the ifg serves as the core region of response inhibition and ifg activity might mirror the attempt to control the bottom - up attention allocation to the tinnitus percept in a top - down manner . thus , the ifg and the ai play important roles in the top - down modulation of automatic or peripheral physiological responses to emotional experiences [ 67 , 68 ] . previous positron emission tomography ( pet ) imaging studies found significant correlation of brain glucose metabolism in the ai and ifg with the distress and/or duration of tinnitus or tinnitus - like sounds [ 6971 ] . taken together , these results suggest that tinnitus distress , salience , or attentional focus is associated with increased intraregional synchronization combined with enhanced functional interconnectivity in these cerebral networks . we also found increased reho values in the smg , a region also associated with the attention network [ 72 , 73 ] . prior eeg or pet studies indicate that the smg may be involved in tinnitus [ 7476 ] . schmidt et al . demonstrated that the dorsal attention network , with seed regions in the bilateral intraparietal sulci , showed decreased correlations with the right smg in tinnitus subjects . thus , the increased local synchronization in the smg of tinnitus patients may alter the connectivity in the dorsal attention network . these features suggested that the alterations of the spontaneous fmri bold signals in attention network might be due to the abnormal local synchronization in chronic tinnitus . by contrast , the left cuneus showed reduced reho values in tinnitus patients , which may be due to compensatory mechanisms in visual regions associated with hearing a phantom sound . it has been suggested that sensory deprivation in the auditory modality could affect the function of the auditory modality [ 77 , 78 ] . we speculated that the decreased reho may be due to increased attention devoted to processing a phantom sound which decreases neural synchronic with the visual system . decreased spontaneous neural activity , as reflected in alff values , was also observed in visual areas , which again could be due to the increased salience of tinnitus . anyhow , it is not appropriate to conclude from the relationships between decreased reho and dysfunction of the visual area in tinnitus . whether reduced reho simply indicates visual impairment in tinnitus is unclear and needs further investigation . we further investigated the interregional synchronized neural activity in tinnitus patients using only seed regions which exhibited high reho values , namely , left and right ai , left ifg , and right smg . the functional connectivity networks from four rois were shown clearly on the three - dimensional brain graphs ( figure 2(c ) ) . remarkably , our study showed significantly enhanced functional connectivity especially within the eca network including the ai , the ifg , and the mfg . the increased functional connectivity between bilateral ai and bilateral mfg was positively correlated with tinnitus distress . many neuroimaging studies have confirmed the involvement of frontal cortex for tinnitus [ 45 , 71 ] . demonstrated structural and functional differences of ventromedial prefrontal cortex in tinnitus patients that were linked to tinnitus loudness , indicating that frontal cortex may contribute to certain perceptual features of tinnitus . the frontoinsular cortex potentially provides executive control overswitching attention between tinnitus and other conditions [ 8082 ] . the differences in tinnitus connectivity might reflect an adaptation to decrease the salience of phantom noises and maintain attention on nonauditory events . burton et al . also confirmed that functional connectivity in areas of eca network in tinnitus group was greater than that in the control group . similar to burton , we also found increased functional connectivity between the ai and the stg , the center of primary auditory cortex . the itg , another temporal cortex related to auditory perception , showed aberrant functional connectivity to the ai and ifg . reported that tinnitus distress was correlated positively with brain metabolism in bilateral itg using pet imaging . in our study , there also existed enhanced interregional synchronization between right smg and left ifg and right ofc , indicating the disrupted attention network associated with chronic tinnitus . besides the attention networks , significantly increased functional connectivity to several dmn regions , such as the precuneus , acc , and pcc , was also observed in tinnitus patients . the dmn , encompassing precuneus and mesiofrontal and temporoparietal junction areas , appears to have strong negative correlations with a network of brain regions commonly activated during the performance of goal - directed cognitive tasks termed the task - positive network . using seed - based , independent component analysis ( ica ) or graph theory analysis , previous functional connectivity studies have found altered interregional synchronization between the dmn and attention network in tinnitus subjects [ 12 , 20 , 84 ] . our study was inconsistent with reduced functional connectivity within the dmn in schmidt 's study as well as the lack of significant differences between the dmn and attention networks in wineland 's study [ 20 , 85 ] . these discrepancies might be due to the methodological features unique to the current study such as the use of seed regions with high reho values , the use of patient with normal hearing up to 16 khz , and elimination of subjects with hyperacusis . this current work was an exploratory study using high reho values to select seed regions for subsequent connectivity analysis and had several inevitable limitations . first , our sample size was moderate , 29 tinnitus patients and 30 normal hearing subjects . increasing the sample size with our approach would increase our ability to make causal relationships between the altered intra- or interregional synchronization and tinnitus characteristics . furthermore , though this study has attempted to minimize the amount of scanner noise with earplugs , we can not completely prevent subjects from hearing some sound . the existence of scanner noise may make the internal sound of tinnitus less salient thereby reducing the differences in resting - state networks between tinnitus and control groups . however , this limitation applies to virtually all resting - state studies in the literatures . nevertheless , this limitation should be taken into consideration while attempting to draw conclusions on resting - state fmri results in studies involving tinnitus patients as well as studies involving auditory stimulation in general . finally , participants with hyperacusis were excluded from the current study because tinnitus is often accompanied by hyperacusis that could cause resting - state pathological brain activation in tinnitus patients according to previous neuroimaging studies [ 86 , 87 ] . since the tinnitus subjects without hyperacusis could not represent all the chronic tinnitus patients , the role of chronic tinnitus accompanying hyperacusis should also be taken into account in future explorations . our combined reho and functional connectivity analyses demonstrated significantly altered infra- and interregional synchronization mainly within the attention network and the dmn prior to structural changes in tinnitus patients . thus , tinnitus can be regarded as the consequence of multiple resting - state networks involved in different aspects of tinnitus , such as auditory perception , attention , and affect . the identification of both local regions of aberrant resting - state activity by reho as well as aberrant interregional disturbances in brain connectivity could help unravel the complex cerebral networks subserving tinnitus and enhance our understanding of the pathophysiological mechanisms of this disorder . he had suffered from intermittent headache for 2 months and had taken benzodiazepines and analgesics under the diagnosis of somatoform disorder at a local clinic . he had a 5-day history of diplopia and a 3-day history of blurred vision that progressed to blindness during the preceding 24 hours . on admission his blood pressure was 130/70 mmhg , pulse rate was 82/min , respiratory rate was 20/min , and body temperature was 36.3. on neurologic examination the patient was alert and exhibited no meningeal signs . only light perception was possible in both eyes , but no other neurological abnormality was noted . lumbar puncture showed the opening pressure of 160 mm h2o with clear cerebrospinal ( csf ) fluid . csf evaluation showed 30 red blood cells / mm , 80 white blood cells / mm , 80% lymphocytes , 41 mg / dl glucose , and 114 mg / dl protein . his body temperature increased to 38.8 1 day after admission , but cbc revealed no leukocytosis . brain mri showed no meningeal enhancement , parenchymal lesion , or intracranial vascular abnormality supplying the optic nerve , optic chiasm , or optic tract . the results of fundoscopic examinations were unremarkable , and visual evoked potential showed no wave formation on bilateral pathways ( fig . an india ink smear of the csf demonstrated encapsulated yeast , and cryptococcus neoformans was cultured ( fig . he was found to be seropositive for hiv with a high antibody titer ( 53.87 s / co ) . he was treated with both antiretroviraland antifungal agents , and his visual acuity had improved upon discharge . neuro - ophthalmic lesions are present in 6% of patients with hiv infection during the course of the disease , with most of them being are attributable to cryptococcal meningitis.3 cryptococcus neoformans , the cause of cryptococcal meningitis , is the fourth most common source of life - threatening infection in aids patients after infections of cytomegalovirus , pneumocystis carinii , and mycobacterium avium intracellulare.1 it is present in pigeon droppings and infects by inhalation of contaminated soil . cryptococcal meningitis is fatal in hiv - infected patients if not treated , and hence early diagnosis is very important . the signs and symptoms of cryptococcal meningitis include headache ( 80~92% of cases ) , meningeal signs ( 50~80% ) , nausea / vomiting ( 40~80% ) , fever ( 36~67% ) , and visual disturbances ( 33~47%).1 our patient presented with acute blindness without other definite clinical symptoms at the time of admission . possible mechanisms for binocular blindness due to cryptococcal meningitis include direct fungal infiltration of the optic nerve , optic chiasm , or optic tracts , adhesive arachnoiditis , cerebral vasculitis , and intracranial hypertension.4 it has been suggested that rapid - onset visual loss is caused by infiltration of the optic nerve or optic chiasm , while slow - onset visual loss is due to increased csf pressure.4 a csf opening pressure exceeding 200 mmh2o and papilledema reflect intracranial hypertension , but our patient showed a normal csf opening pressure and unremarkable fundoscopic examination findings . moreover , his visual symptoms developed very early in the course of the disease . thus , the sudden visual loss might have been due to retrobulbar fungal infiltration . whereas the prevalence of cryptococcosis is decreasing because of the widespread availability of antiretroviral therapy , cryptococcal meningitis is still a fatal complication of hiv infection . thus , both early diagnosis of cryptococcal meningitis and detection of the underlying causes are important . in our opinion , unexpected sudden binocular blindness should be considered as a possible initial manifestation of cryptococcal meningitis related to hiv infection .

objective . subjective tinnitus is hypothesized to arise from aberrant neural activity ; however , its neural bases are poorly understood . to identify aberrant neural networks involved in chronic tinnitus , we compared the resting - state functional magnetic resonance imaging ( fmri ) patterns of tinnitus patients and healthy controls . materials and methods . resting - state fmri measurements were obtained from a group of chronic tinnitus patients ( n = 29 ) with normal hearing and well - matched healthy controls ( n = 30 ) . regional homogeneity ( reho ) analysis and functional connectivity analysis were used to identify abnormal brain activity ; these abnormalities were compared to tinnitus distress . results . relative to healthy controls , tinnitus patients had significant greater reho values in several brain regions including the bilateral anterior insula ( ai ) , left inferior frontal gyrus , and right supramarginal gyrus . furthermore , the left ai showed enhanced functional connectivity with the left middle frontal gyrus ( mfg ) , while the right ai had enhanced functional connectivity with the right mfg ; these measures were positively correlated with tinnitus handicap questionnaires ( r = 0.459 , p = 0.012 and r = 0.479 , p = 0.009 , resp . ) . conclusions . chronic tinnitus patients showed abnormal intra- and interregional synchronization in several resting - state cerebral networks ; these abnormalities were correlated with clinical tinnitus distress . these results suggest that tinnitus distress is exacerbated by attention networks that focus on internally generated phantom sounds . ocular complications of hiv - related cryptococcal meningitis are reasonably common , but complete binocular blindness as the first manifestation of hiv is extremely rare . a 58-year - old man presented with binocular blindness . he experienced blurred vision for 3 days before the blindness . mild pleocytosis was present in the cerebrospinal fluid , from which cryptococcus neoformans was cultured . serology revealed positivity for hiv antibody . he was treated with antifungal and antiretroviral therapy . this case indicates that hiv - related cryptococcal meningitis should be taken into consideration when determining the cause of unexpected sudden binocular blindness .

nonalcoholic fatty liver disease ( nafld ) is the most common chronic liver disease in europe and the us with a prevalence of up to 30% . it is associated with obesity and diabetes and is thus considered to be the hepatic manifestation of the metabolic syndrome . nafld ranges from simple hepatic steatosis , characterized by triglyceride accumulation in hepatocytes , to nonalcoholic steatohepatitis ( nash ) , which may be associated with fibrosis and progression to cirrhosis or hepatocellular carcinoma . while the diagnosis of nafld is based on histology and patients history , the increase in prevalence demands noninvasive methods for diagnosis and surveillance . hepatocyte apoptosis was identified as a key feature of nafld and correlates with disease severity . cytokeratin 18 ( ck18 ) is an intermediate filament expressed in single - layer epithelial tissues . during apoptosis , caspase - cleaved ck18 is released into the cytoplasm and released into the serum after cell death . therefore , soluble forms of extracellular ck18 in the serum are utilized to quantify activity of cell death . wieckowska et al . quantified cleaved ck18 fragments in patients with nafld and found a correlation with the occurrence of liver fibrosis and hepatic inflammation . [ 5 , 6 ] the published results of a multicenter validation study by feldstein et al . revealed ck18 fragments as a predictor of nash versus simple steatosis . most of its disassembly takes place in endothelial liver cells . because of the lack of function , fibrosis and cirrhosis lead to impaired clearance of hyaluronic fragments . several studies could show a correlation between serum hyaluronic acid and fibrosis stage in chronic liver diseases , including nafld [ 8 , 9 ] . here , we evaluated caspase - cleaved ck18 and hyaluronic acid as biomarkers for nafld and fibrosis in a cohort of 127 patients who underwent bariatric surgery and compared these results to the histological diagnosis , quantified by nafld - activity score ( nas ) , as established by kleiner et al . . in line with recent publications , we observed a clear correlation between caspase - cleaved ck18 and liver steatosis . a total of 127 morbidly obese patients ( mean age : 45 10 ; 78.7% females , mean bmi : 52 8) who underwent bariatric surgery at a center for bariatric surgery were included . indication for bariatric surgery was made according to national institutes of health ( nih ) guidelines ( bmi 40 kg / m or 35 kg / m , plus comorbidities ) . subjects reporting excess alcohol consumption ( > 20 g / day in males or > 10 g / day in females ) indicating alcoholic liver disease were excluded . the surgeon 's choice that is , adjustable gastric band , roux - y , or gastric bypass surgery was based on the current guidelines as adapted to the patient 's clinical conditions and comorbidities as well as on clinical experience . a control group of 10 healthy volunteers ( 7 males ; 3 females / median age : 26 7.6 years ) had an average bmi of 22.4 0.82 kg / m . this study was approved by the ethics committee ( institutional review board ) of the university hospital essen . patients volunteering were informed about intraoperative risks and benefits of wedge liver biopsy and provided informed consent . ck18 serum levels and hyaluronic acid were assessed in the sera of patients and healthy controls using the m30-apoptosense ( peviva , bromma , sweden ) and hyaluronic acid ( wako chemicals , neuss , germany ) elisa kits . the m30-apoptosense assay detects a specific epitope of caspase - cleaved ck18 , which will further be referred to as m30 . patients with and without nash were compared regarding metabolic characteristics , transaminases , nas , m30 , and hyaluronic acid levels using independent samples t - tests ( mann - whitney test ) . medline research was performed in january 2011 with the search terms serum markers nafld . analyses were performed with with spss 15.0.1 , version 2006 ( spss , chicago , il , usa ) and graph pad , version 5.03 ( graph pad , graph pad software inc . , ca , usa ) . the clinical characteristics of the 56 patients with nash ( nas 5 ) in comparison to 71 nafl patients ( nas < 5 ) are stated in table 1 . in patients with nash , risk factors of the metabolic syndrome as triglycerides and fasting glucose were higher in comparison to patients with nafl . as the nas score consists of the individual scoring values for steatosis , ballooning , and lobular inflammation , a nas 5 was accompanied with significantly higher scores for the individual characteristics of nafld . as expected , m30 was elevated in nash patients ( figures 1(a ) , 2(a ) , and 2(b ) ) . accordingly , serum transaminase levels were elevated in nash patients ( figure 1(b ) ) . the artificial cut - off for m30 to predict nash ( 275 u / l ) , established and validated by the feldstein group , in our cohort corresponded with histological steatosis , but not lobular inflammation . in serological nash , ballooning and nas score were only modestly elevated ( figure 1(c ) ) . however , alanine aminotransferase ( alt ) and aspartate aminotransferase ( ast ) levels were significantly higher in patients with plasma levels of m30 275 u / l , indicating hepatocellular damage , despite no histological signs of lobular inflammation ( figure 1(d ) ) . interestingly , about two - third of our cohort had stage-2 fibrosis ( figures 2(a ) and 2(b ) ) . we did not find an increase in fibrosis score in patients with a nas 5 . however , morphometrically quantified liver collagen ( sirius red ) and serum hyaluronic acid were both elevated in histological nash versus nafl ( figures 3(a ) , 2(e ) , and 2(f ) ) . advanced fibrosis was further associated with more tunel positive cells per visual field in liver sections , and serum hyaluronic acid was dramatically increased in patients with advanced fibrosis ( figures 3(b ) , 2(c ) , and 2(d ) ) . accordingly , m30 levels were higher in patients with advanced fibrosis , while the nas score was not different between the groups at all ( figure 3(c ) ) . this supports the above - mentioned observation that fibrosis is associated with hepatocellular apoptosis , rather than lobular inflammation . in this context , the number of tunel positive cells in livers was significantly correlated with the percentage of sirius red positive area in the visual field and m30 was significantly correlated with serum hyaluronic acid ( figure 3(e ) ) . in our cohort of 127 morbidly obese patients , caspase - cleaved ck18 correlated with hepatic steatosis but not lobular inflammation as assessed by histological scoring . still , ast and alt were significantly higher in patients with ck18 levels above the established cut - off for nash , indicating a discrepancy between histological assessed inflammation and actual liver injury . interestingly , the degree of fibrosis did not correspond to the nas score , while a high apoptosis rate , as assessed by tunel staining in liver tissue and assessment of serum caspase - cleaved ck18 , correlated with increased fibrosis . this is in line with recent publications , that hepatocyte apoptosis , rather than hepatic inflammation , is a key trigger of hepatic stellate cell activation and thus fibrogenesis [ 12 , 13 ] . taken together , our data indicate that biomarkers in nafld might reveal more information than standard histological scoring . in the following , we will review the emerging data on serum markers in nafld , a field that is continuously growing ( figure 3(f ) ) . to date , liver biopsy and histological assessment of liver steatosis , ballooning , and lobular inflammation in h&e staining remain the gold standard for diagnosis of nash . however , despite a low rate of overall complications , percutaneous liver biopsy remains an invasive procedure with the risk of potentially lethal hemorrhage and infections and is further complicated in the growing number of obese patients with nafld [ 14 , 15 ] . given the increasing prevalence of nafld , noninvasive markers for nafld are a promising approach not only for screening reasons . in 2006 , wickowska et al . quantified soluble caspase - cleaved ck18 with a specific m30-elisa and found a correlation with the histological staging in nafld patients . this was validated by the same group and in a cohort of patients who underwent bariatric surgery , an m30-cutoff value of 275 u / l was established to discriminate simple steatosis from nash [ 7 , 17 ] . we and others were able to show that m30 correlates with fatty acid transporter expression in liver tissue and specific fatty acids in the serum [ 18 , 19 ] . in a cohort of patients with polycystic ovarial syndrome , we could identify a high rate of patients with nash utilizing this assay . lately , the feldstein group enhanced sensitivity and specificity of nash detection by detection of soluble fas and fas ligand additional to m30 and established an apoptotic index in the noninvasive diagnosis of nash . as mentioned above , we found not only a correlation of m30 with hepatic steatosis but also a good correlation between the apoptosis rate and markers for fibrosis in our cohort , which is in line with observations from other groups . hepatocyte apoptosis is known to activate hepatic stellate cells via formation of apoptotic bodies , which are either directly stimulating hepatic stellate cells or are engulfed by kupffer cells , which in turn activate stellate cells [ 23 , 24 ] . noninvasive diagnosis of fibrosis in nafld is complicated by the high rate of obese patients compared to hepatitis c infection . transient elastography , a method that accurately predicts fibrosis stage in hepatitis c patients , faces limitations in nash patients , as accuracy is dramatically reduced in obese individuals . hyaluronic acid has been implied as a good biomarker for fibrosis and was correlated with the apoptosis rate in our cohort . however , several multipanel tests are among the most promising in noninvasive diagnosis of fibrosis . these tests consist of different serum parameters , some even include assessment of hyaluronic acid and are validated in cohorts of hepatitis c patients but might as well be useful in patients with nafld , although validation studies still need to prove accuracy in this cohort [ 26 , 27 ] . taken together , because of the nature of nafld and the increase in prevalence , there is an increasing demand for noninvasive markers of nafld and fibrosis . since hepatocyte apoptosis is a key feature of nafld and also contributes to fibrogenesis , assessment of soluble markers of apoptosis appears to be a good alternative to liver biopsy in these patients . in fact , these methods might give better insight into the status and prognosis of liver disease than standard h&e assessment . however , especially fibrotic markers need further validation since the high rate of obesity might confound these tests . drug - induced gingival overgrowth ( digo ) remains a significant problem for the dental clinicians . amlodipine is a third - generation dihydropyridine calcium channel blockers ( ccb ) that is used in the management of both hypertension and angina . ellis et al . , first reported gingival sequestration of amlodipine and amlodipine - induced gingival overgrowth ( aigo ) . since then , very few isolated cases of aigo have appeared in the dental literature although there are numerous reports of nifedipine induced gingival overgrowth until date . the incidence of gingival hypertrophy with nifedipine therapy has been reported to be as high as 20% . other synonyms have been used to describe similar lesions , such as inflammatory pseudotumor , histiocytoma , xanthomatous granuloma , inflammatory myofibroblastic tumor , and spindle cell pseudotumor . it occurs primarily in the lungs , but has occurred in other extra - pulmonary sites . this lesion is not a neoplastic process , nor is it associated with a monoclonal expansion of a single plasma cell instead ; this is a reactive , inflammatory lesion which usually involves the mobile tissues of the oro - nasopharyngeal region , such as paranasal sinuses , buccal mucosa , tongue , and lip . they are even rarer on the gingiva and very few case reports have been documented . there are no reports of amlodipine - related plasma cell granuloma of gingiva existing in the extant literature . the present case report describes an unusual case of amlodipine induced massive plasma cell granuloma on the gingiva . a 54-year - old female patient reported to the multispecialty private dental clinic ( latur , india ) with the chief complaint of diffuse swelling in the right maxillary anterior region . patient was not aware of such growth until 8 months back when she noticed a small slowly growing bead - like nodular enlargement of the gums that gradually progressed to the present size covering almost the entire front teeth . her past medical history revealed that the patient was hypertensive for last 2 years and was under medication ( amlodipine 10 mg , once daily ) . the lesion was asymptomatic , but the patient complained it to be severely interfering with mastication , speech , and oral hygiene practice resulting in functional and aesthetic problem . on intraoral examination , the lesion was a well - circumscribed exophytic sessile spherical mass of 1.5 inches diameter with color same as that of the surrounding oral mucosa with the scattered erythema [ figure 1 ] . the lesion was extended from distal surface of upper right canine to distal surface of upper left central incisor crossing the midline . poor oral hygiene status of the patient was assessed from the presence of local irritating factors contributing to the mild inflammatory component of the gingival enlargement . pre - operative clinical presentation of amlodipine induced plasma cell granuloma of gingiva complete hemogram showed all blood counts to be within the normal limits . intraoral periapical radiograph and orthopantomogram in the region of aigo showed generalized advanced horizontal bone loss around all teeth resulting in pathologic migration the lesion was biopsied under local anesthesia . the area was sutured and the specimen submitted for histopathological examination . as the extraction of all the remaining teeth with poor prognosis was planned substitution of amlodipine was not considered . planned extraction of remaining mandibular teeth was carried out in subsequent appointment . following healing period of 2 months complete denture prosthesis histopathological examination using hematoxylin and eosin stain , revealed proliferative parakeratinized stratified squamous epithelium , connective tissue with sheets of plasma cells intermixed with scattered small lymphocytes . high power microscopy : h and e staining shows sheets of plasma cells intermixed with scattered small lymphocytes immunohistochemical study of the biopsy material revealed the polyclonal plasma cell infiltrate uniformly positive for cd138 , a marker for plasmacytoid cells [ figure 3 ] and kappa light chain [ figure 4 ] and weak expression was noted for the lambda light chain . absence of findings from common tetrad of multiple myeloma ( crab : c = calcium ( elevated ) , r = renal failure , a = anemia , b = bone lesions ) ruled out its possibility . positive staining for d138 positive staining for kappa light chain on the basis of clinicohistopathologic examination and immunohistochemistry , a confirmative diagnosis of plasma cell granuloma was made . healing was uneventful and the patient is presently under follow - up since 5 months . gingival overgrowth is an often overlooked , but potentially harmful side - effect of treatment with amlodipine and other ccb . the reason for this adverse event is not absolutely known , but two main inflammatory and non - inflammatory pathways have already been suggested . the proposed non - inflammatory mechanisms include defective collagenase activity due to decreased uptake of folic acid , blockage of aldosterone synthesis in the adrenal cortex and consequent feedback increase in adrenocorticotropic hormone level , and up - regulation of keratinocyte growth factor . alternatively , inflammation may develop as a result of direct toxic effects of concentrated drug in gingival crevicular fluid and/or bacterial plaques . this inflammation could lead to the up - regulation of several cytokine factors such as transforming growth factor beta . in a series of 150 cardiac patients , it was found that amlodipine at a dose of 5 mg / day can not induce gingival hyperplasia even if taken more than 6 months . plasma cells are terminally differentiated b lymphocytes , which are typically found in the red pulp of the spleen , medulla of the lymph nodes , tonsils , lamina propria of the entire gastrointestinal tract , mucosa of the nose and upper airway , and sites of inflammation . a plasma cell 's main function is to produce immunoglobulins or antibodies . periodontal lesions with a predominance of plasma cells were reported initially by during late 1960s and only very few case reports have been documented since then . these cases reported a similar reactive gingival growth with similar histological and clinical appearance , which was treated by excisional biopsy , as in the present case . gingival plasma cell granulomas have been reported in patients with cyclosporine - induced gingival overgrowth . it was suggested that interleukin-6 and phospholipase c-1 may induce heavy plasma cell infiltration in cyclosporine - induced gingival overgrowth . it is important to differentiate plasma cell granulomas from tumors of bone such as multiple myeloma , solitary myeloma , and soft - tissue myeloma ( extramedullary plasmacytoma ) considering the poor prognosis of these neoplasms . differentiating the type of soft - tissue tumor is mandatory , as plasma cell granuloma may be benign , extramedullary plasmacytoma may be malignant or a precursor to malignancy . histologically , extramedullary plasmacytoma consists of mixture of typical and atypical plasma cells while plasma cell granuloma consists of normal plasma cells and small lymphocytes that are surrounded by connective tissue septa . the immunohistochemical analysis have shown that in the case of malignancy ratio of the kappa to lambda light chain may be greater than 10:1 or 1:10 , whereas in a reactive lesion the ratio is 2:1 . plasma cell lesions with the predominance of plasma cells may represent an autoimmune reaction or an alteration of blood - flow imposing congestive vasodilatation or lesions occurring due to parasitic infiltration . recent studies have reported that a gingival plasma cell granuloma shows variable gene expression for cell - mediated immunity and stromal tissue degeneration , undergoing sclerotic fibrosis with a persistent inflammatory reaction . in conclusion , amlodipine induced plasma cell granuloma of gingiva is a distinct pathological entity characterized by the presence of mature polyclonal plasma cell sheets and fibrovascular connective tissue stroma . while the mechanism of amlodipine - induced gingival plasma cell granuloma of gingiva is considered to be multi - factorial , the drug / cellular interaction may play an important role in the pathogenesis of this entity . this case highlights the need to biopsy unusual lesions to rule out potential neoplasms and also emphasizes the need to submit the excised tissue for histopathological examination regardless of clinical notion and/or perceived surgical success .

hepatocyte apoptosis is a key event in nonalcoholic fatty liver disease ( nafld ) , and serum apoptotic markers are emerging as surrogate markers for nafld . we studied the role of caspase - cleaved cytokeratin18 in the diagnosis of fibrosis in a cohort of 127 morbidly obese patients and also performed a review of the literature biomarkers of nafld and fibrosis . here , we found that cleaved caspase 18 correlated with liver steatosis and liver injury as assessed by serum transaminase levels . furthermore , hepatocyte apoptosis as assessed by cleaved ck18 and tunel staining correlated with the extent of fibrosis as assessed by sirius red staining and serum hyaluronic acid . these results underscore the important role of hepatocyte apoptosis in the pathogenesis of fibrosis in nafld , which led to the utilization of surrogate markers for apoptosis in the noninvasive diagnosis of nafld . we furthermore reviewed current literature of biomarkers of nafld and fibrosis . drug - induced gingival overgrowth ( digo ) can be a serious concern for both patients and clinicians . digo is a well - documented side - effect of some pharmacologic agents , including , but not limited to , calcium channel blockers , phenytoin , and cyclosporine . plasma cell granulomas ( pseudotumors ) are exceedingly rare , non - neoplastic , reactive tumor - like proliferation , primarily composed of plasma cells that manifest primarily in the lungs , but may occur in various anatomic locations . intraoral plasma cell granulomas involving the lip , oral mucosa , tongue , and gingiva have been reported in the past . this is the first case report of amlodipine induced plasma cell granuloma of the gingiva in the medical literature presenting a 54 year - old female patient with hypertension , who received amlodipine ( 10 mg / day , single dose orally ) for 2 years , sought medical attention because of developing maxillary anterior massive gingival overgrowth causing functional and esthetic problem , which was treated by excisional biopsy . histologically , these lesions were composed of mature plasma cells , showing polyclonality for both lambda and kappa light chains and fibrovascular connective tissue stroma confirming a diagnosis of plasma cell granuloma . this case also highlights the need to biopsy for unusual lesions to rule out potential neoplasms .

ludwig 's angina is a known , yet a rare surgical emergency that is potentially life threatening unless early recognised and aggressively treated . we highlight these controversies with a brief review of the literature and a retrospective review of recent experiences with ludwig 's angina . a 56-year - old male presented with a 72-hour history of a worsening dysphagia and submandibular swelling . the onset was following a visit to the dentist where a dental abscess was drained . 24 hours later he revisited his dentist complaining of a submandibular swelling , and was commenced on oral co - amoxiclav . despite that , his symptoms progressed and he made his way to our facility 72 hours later . based on his history and symptoms the diagnosis of ludwig 's angina was suspected . he was not in respiratory distress ; his respiratory rate was 23 , and o2 saturation was 96% on room air . based on this initial assessment , the decision was to manage him conservatively with close airway observation . he was commenced on intravenous co - amoxiclav and clindamycin , and a contrast - enhanced ct scan of his neck and upper thorax was performed to rule out the possibility of a deep neck abscess . this showed an extensive submandibular soft tissue swelling and inflammation with moderate tongue elevation and posterior displacement causing a degree of oropharyngeal airway compromise . no abscess cavity or laryngeal airway compromise was evident . he was admitted to the ear , nose and throat ( ent ) ward for hourly airway observation and intravenous antibiotic treatment . his symptoms improved on a daily basis maintaining a normal breathing rate and pattern with no o2 desaturations below 97% on room air on pulse oximetry . by the 5th day following his admission , his symptoms have fully resolved , and he was discharged from hospital on oral co - amoxiclav . two weeks and 3 months out - patient department followups showed no recurrent soft tissue swelling or oedema , and he was discharged from our service . a 59-year - old male with a background history of noninsulin - dependant diabetes mellitus and sebopsoriasis presented with 24-hour history of mild right - sided facial swelling , a worsening submandibular swelling , and mild dysphagia . he had low - grade pyrexia of 37.8c , his respiratory rate was within normal limits , and his o2 saturation was 98% on room air . on physical examination he had a mild right - sided facial swelling , a significant submandibular swelling , and skin erythema extending inferiorly to the level of the upper border of the sternum . he was commenced on intravenous clindamycin , ciprofloxacin , and benzylpenicillin , and a contrast - enhanced ct scan of his neck and upper thorax ( figures 1 and 2 ) was performed to role out a deep neck abscess or a mediastinum extension . this showed a significant submandibular soft tissue inflammation and oedema with a degree of oropharyngeal compromise . his laryngeal airway was normal , and no deep neck abscess or fascial extension was evident . he was admitted to the ent ward for hourly airway observation and intravenous antibiotic treatment . on the third day after admission , on the other hand , a worrying inferiorly spreading skin erythema to the level of the 5th costal cartilage was noted . despite that , his respiratory rate and o2 saturation remained unchanged , and he had no cardiothoracic complaints to suggest a danger space extension or a necrotising fasciitis . a clinical assessment including a chest plain film and a neck ultrasound was performed . the dermatology service was consulted , and a secondary psoriatic flare up was suspected . a topical daktacort hydrocortisone cream ( miconazole and hydrocortisone ) was added . a gradual improvement of his symptoms occurred on daily basis . by the sixth day following his admission his symptoms two weeks and 3 months out - patient department followups showed no recurrent laryngeal oedema or skin erythema , and he was discharged from our service . ludwig 's angina is named after the german physician , wilhelm friedrich von ludwig who first described this condition in 1836 . it is a potentially life - threatening cellulitis , or connective tissue infection , of the neck and floor of the mouth which is characterised by progressive submandibular swelling with elevation and posterior displacement of the tongue [ 2 , 3 ] . early antibiotic treatment should be broad spectrum to cover gram - positive and gram - negative bacteria as well as anaerobes . the use of intravenous steroids has been proposed as a mean of reducing soft tissue swelling and oedema and minimising the likelihood for the need of a surgical airway in ludwig 's angina [ 1 , 5 , 6 ] . this remains controversial , as up to this date no randomised controlled trials that demonstrate the efficacy of corticosteroids in these patients exist . traditionally aggressive airway management by securing the airway with endotracheal intubation or surgically with a surgical tracheostomy was the norm . although no specific guidelines are present for managing acute ludwig 's angina , decisions regarding airway protection are largely dependant on the practice guidelines for management of the difficult airway that were adopted by the american society of anaesthesiologists in 1992 and updated in 2003 . in these guidelines , a difficult airway is defined as the clinical situation in which a conventionally trained anaesthesiologist experiences difficulty with face mask ventilation of the upper airway , difficulty with tracheal intubation , or both . the guidelines specify that these recommendations may be adopted , modified , or rejected according to the clinical needs and constraints as these guidelines are not intended as standards or absolute requirements and their purpose is to assist the practitioner in decisions about health care . recent reports have encouraged conservative management of ludwig 's angina in selected patients over the conventional aggressive airway management . larawin et al . retrospectively studied a total of 103 patients with deep neck space infections from 1993 to 2005 . ludwig 's angina was the most commonly encountered infection seen in 38 ( 37% ) patients of treatment . 13 ( 34% ) patients managed successfully with medical therapy and only 4 ( 10% ) patients required a tracheostomy tube . kurien et al . reported a 13-year review of patients with ludwig 's angina between 1982 and 1995 . 70% were controlled with conservative medical management while 81% of adults required incision and drainage . a 9-year review by greenberg et al . of 29 cases of deep neck space infections reported 21 patients ( 72 % ) treated conservatively following initial clinical assessment . of those treated nonconservatively at initial presentation , 7 ( 24% ) patients were able to be intubated using fiberoptic nasoendoscopy and 1(3% ) patient required tracheostomy under local anaesthesia . early detailed imaging is essential to evaluate the extension of tissue infection or necrosis and to guide decisions regarding surgical approaches when indicated . ct scan and mri are of invaluable importance in the assessment of deep neck space infections and collections . miller et al . reported that combined clinical evaluation and ct findings lead to accuracy of 89% , sensitivity of 95% , and specificity of 80 % in identifying drainable collection . plain chest radiographs are useful when looking for signs of mediastinum extension such as mediastinitis and pleural effusion . although ultrasound is not as easily interpreted by clinicians and surgeons as other imaging modalities , its availability , cost effectiveness , reduced risk of radiation , and accuracy in differentiating cellulites - related oedemas from abscess collections make it a reliable supplement modality to ct scan in resistant cases [ 12 , 13 ] . however , in cases of significant airway compromise where an immediate decision regarding the need of a definitive airway is required , clinical experience and judgment are superior to imaging . from our clinical experience and the literature review we conclude that conservative management of ludwig 's angina is acceptable in selective cases , provided that early antibiotic therapy is commenced and any collectable abscess is drained . initial airway assessment is based on respiratory rate , oxygen saturation , and findings on fiberoptic laryngoscopy . patients are then categorised as having either a severe airway compromise or a stable airway . in the severely compromised group ( patients unable to maintain saturation on room air above 95% , respiratory rate > 25 , or awake fiberoptic - assisted intubation should be attempted first ; if this fails then a surgical tracheostomy is performed under local anaesthesia . in the other group , where patients are able to maintain normal oxygen saturation and respiratory rate on room air and where no significant airway compromise is evident on fiberoptic examination , this involves close airway observation ( oxygen saturation , respiratory rate , and serial fiberoptic laryngoscopy ) in a high dependency unit ( hdu ) or ent ward . after the initial clinical assessment and airway decision all patients should undergo ct scanning of their neck and thorax for further detailed airway and deep neck spaces evaluation . any abscess or collection cavity should be drained , and both groups should be kept in an hdu or ent ward for hourly airway assessment for 2448 hours . this is based on close airway observation on a specialised airway unit and a serial clinical airway assessment . improved imaging modalities , antibiotic therapy , surgical skills , and clinical experience are the key factors behind this change in practice . 62 newly diagnosed cases of moderate to severe primary glaucoma were recruited from the out - patient department and glaucoma clinics of a tertiary care center in new delhi , india . patients with characteristic changes in the optic nerve head and corresponding glaucomatous visual field defects on standard automated perimetry were included in the study . all patients had best corrected visual acuity of at least 20/200 in the better eye , were over 40 years of age and were conversant in hindi or english . exclusion criteria included subjects with history of previous treatment for glaucoma ; vision < 20/200 in better eye ; presence of other comorbid ocular conditions , e.g. , age - related macular degeneration , cataract , optic neuropathy other than glaucoma , that could potentially contribute to visual loss ; chronic systemic diseases that could significantly affect qol , e.g. , diabetes , arthritis , coronary artery diseases , cerebrovascular diseases , collagen vascular disorders , history of any ocular surgery in previous 3 months ; deaf or communication impaired ; and physically disabled individuals . eligible patients underwent a thorough history and ocular examination including evaluation of visual acuity ; slit lamp biomicroscopy , intraocular pressure ( iop ) assessment with goldmann applanation tonometer ; gonioscopy using goldmann two - mirror gonioscope ; and optic nerve head evaluation using a 90 d lens . visual fields examination was performed using the humphrey visual field analyzer ( humphrey instruments inc . , allergan humphrey , san leandro , ca ) using the swedish interactive threshold algorithm 302 . the patients were categorized with respect to the severity of glaucoma according to the hodapp parrish anderson classification . sixty healthy control subjects with no ocular or systemic disease visiting the hospital for refractive error were included . the controls were age - matched with the glaucoma cases and were evaluated in a similar manner . the ind vfq33 is a 33-item questionnaire comprising of 33 items in three domains ( annexure ) . the general functioning elicited items related to distance vision , near vision , ambulation , night vision , dark adaptation , color vision , depth perception . the psychosocial impact domain related to the feelings of the patient related to low vision , e.g. , whether the subject feared going out at night , was he / she a burden on their family or fear of losing the remaining vision . finally , visual symptoms domain described most common visual symptoms like glare , discomfort , and blurring . general functioning ( domain 1 ) had 21 items ( q1-q21 ) , a range of response varied from 1 to 5 giving a total range for domain 1 ( 21105 ) . psychosocial impact ( domain 2 ) and visual symptoms ( domain 3 ) had 5 items ( q22-q26 ) and 7 items ( q27-q33 ) , respectively . domain 2 and domain 3 had 14 as the range of response with a total score of responses varying from 5 to 20 in psychosocial impact and 128 in visual symptoms domain . the summary scores from each domain were added to give a total qol score with a minimum score of 33 and maximum 153 . the higher the score , the worse the qol . the mean total qol score of controls and glaucoma patients at each visit was divided into quartiles viz . no difficulty ( < 25% ) , mild difficulty ( 25% 50% ) , moderate difficulty ( > 5075% ) and severe difficulty ( > 75% ) with cut - off score of 3338 , 3844 , 4473 and > 73 respectively . the questions were translated and back - translated to the native language , hindi , for standardization . the ind - vfq33 questionnaire across the three domains was administered to the subjects , by a single investigator ( va ) in the native language . the evaluation included ophthalmological work - up including visual acuity assessment , slit lamp biomicroscopy , measurement of iop , optic disc assessment and repeat visual field testing using the same algorithm . qol assessment was repeated using ind - vfq33 questionnaire , this being administered by the same investigator ( va ) in same language as done at the initial visit . the responses were entered into microsoft excel 2007 data sheet and statistical analysis was done using stata 11 19852011 statacorp lp ( stata press , texas , usa ) . the mean qol scores of glaucoma patients obtained were compared with that of controls using the unpaired t - test . multiple linear regression was used to assess significant factors affecting the qol at baseline and 3 months after treatment . factors like age , sex , visual acuity in the better eye , iop in the better eye , glaucoma severity and number of medications used were included as these may affect the qol of the patient . correlation values between visual acuity in the better eye , presenting iop , glaucoma severity and total qol score were obtained at baseline and at 3 months . the ind vfq33 is a 33-item questionnaire comprising of 33 items in three domains ( annexure ) . the general functioning elicited items related to distance vision , near vision , ambulation , night vision , dark adaptation , color vision , depth perception . the psychosocial impact domain related to the feelings of the patient related to low vision , e.g. , whether the subject feared going out at night , was he / she a burden on their family or fear of losing the remaining vision . finally , visual symptoms domain described most common visual symptoms like glare , discomfort , and blurring . general functioning ( domain 1 ) had 21 items ( q1-q21 ) , a range of response varied from 1 to 5 giving a total range for domain 1 ( 21105 ) . psychosocial impact ( domain 2 ) and visual symptoms ( domain 3 ) had 5 items ( q22-q26 ) and 7 items ( q27-q33 ) , respectively . domain 2 and domain 3 had 14 as the range of response with a total score of responses varying from 5 to 20 in psychosocial impact and 128 in visual symptoms domain . the summary scores from each domain were added to give a total qol score with a minimum score of 33 and maximum 153 . the higher the score , the worse the qol . the mean total qol score of controls and glaucoma patients at each visit was divided into quartiles viz . no difficulty ( < 25% ) , mild difficulty ( 25% 50% ) , moderate difficulty ( > 5075% ) and severe difficulty ( > 75% ) with cut - off score of 3338 , 3844 , 4473 and > 73 respectively . the questions were translated and back - translated to the native language , hindi , for standardization . the ind - vfq33 questionnaire across the three domains was administered to the subjects , by a single investigator ( va ) in the native language . patients were reassessed at 3 months following the start of the ocular hypotensive therapy . the evaluation included ophthalmological work - up including visual acuity assessment , slit lamp biomicroscopy , measurement of iop , optic disc assessment and repeat visual field testing using the same algorithm . qol assessment was repeated using ind - vfq33 questionnaire , this being administered by the same investigator ( va ) in same language as done at the initial visit . the responses were entered into microsoft excel 2007 data sheet and statistical analysis was done using stata 11 19852011 statacorp lp ( stata press , texas , usa ) . the mean qol scores of glaucoma patients obtained were compared with that of controls using the unpaired t - test . multiple linear regression was used to assess significant factors affecting the qol at baseline and 3 months after treatment . factors like age , sex , visual acuity in the better eye , iop in the better eye , glaucoma severity and number of medications used were included as these may affect the qol of the patient . correlation values between visual acuity in the better eye , presenting iop , glaucoma severity and total qol score were obtained at baseline and at 3 months . the glaucoma subjects and the control population were well matched for age , gender and literacy rates ( p = 0.71 , 0.91 and 0.18 respectively ) . better eye logmar visual acuity was 0.35 0.25 and 0.08 0.11 in glaucoma patients and controls respectively ( p < 0.001 ) . glaucoma subjects had significantly higher baseline iop in the better eye than the control population ( 25.4 6.6 vs. 11.6 2.1 mm of hg respectively , p < 0.001 ) . demographic characteristics of glaucoma patients and controls a comparison of ind - vfq33 total and domain wise scores is provided in table 2 . there was a significant difference between the two groups with respect to the mean total qol scores , general functioning domain , psychosocial impact and visual symptoms ( p < 0.001 ) . as shown in table 3 , the total mean score increased from 74.8 to 78.3 ( p < 0.001 ) on repeating the assessment 3 months after initiating the anti - glaucoma therapy suggesting worsening of qol . comparison of baseline qol scores in glaucoma patients and controls qol scores of glaucoma patients at baseline and 3 months multiple linear regression analysis was used to assess the effect of various factors on the qol as assessed with the ind - vfq33 questionnaire [ table 4 ] . better eye visual acuity was found to have a significant contribution to the qol scores , both at baseline and after 3 months of starting ocular hypotensive therapy ( p < 0.001 and < 0.05 respectively ) . it was also noted that the use of more than two ocular hypotensive medications was significantly related to poor qol at 3 months assessment ( p < 0.05 ) . multiple regression analysis showing the correlation of factors and qol among glaucoma subjects table 5 gives a list of items and response scores that were significantly different between the glaucoma and control subjects . the general functioning domain showed that most of the glaucoma patients had significant difficulty in ambulation , night vision , recognition , and dark adaptation as compared to controls . most glaucoma patients were frightened to go out at night and were worried that they may lose their remaining vision . problems of glare and low vision were also significantly higher in glaucoma patients than controls . a comparison of commonly reported problems in glaucoma patients and their statistical significance as compared to control subjects table 6 elaborates the patient parameters and qol scores across the primary open - angle glaucoma ( poag ) and primary angle closure glaucoma ( pacg ) population as included in the study . these groups differed significantly in the visual acuity at baseline and at 3 months , being worse in the pacg population ( p < 0.001 ) . the mean iop and mean glaucoma severity as seen by the visual field mean deviation scores were comparable across the two groups . pacg patients had worse qol scores than the poag population both at baseline and at 3 months after initiating ocular hypotensive medications ( p = 0.12 and 0.09 respectively ) , however , the difference did not reach statistical significance . this is the first study that evaluates the vision related qol in newly diagnosed glaucoma patients using a questionnaire developed and validated specifically for the population of the indian subcontinent . have earlier validated this questionnaire for vision related qol in 273 cataract patients in southern parts of india . they noted that patients with severe visual impairment and blindness reported significantly worse vision - specific mobility and activity limitation ( mean change 18.82 , p = 0.007 and 29.48 , p < 0.001 respectively ) compared to those with no visual impairment . nelson et al . have developed a glaucoma specific gql-15 scale , and defined the vision related problems perceived by glaucoma patients , e.g. , peripheral field , color vision , glare , dark adaptation , which are well covered in the ind - vfq33 questionnaire . this study shows that subjects with glaucoma had worse qol as compared to control population . the fact that this further worsened after initiation of medical therapy has some pertinent implications . this highlights that the diagnosis of glaucoma , a chronic sight - threatening condition that may require life - long therapy , may have a significant psychological impact on a patient . this should be identified , acknowledged and managed by appropriate disease - related education and counseling . our results are similar to those reported by nelson et al . where they have noted a statistically significant decrease in performance - related qol between normal subjects and all groups of glaucoma patients . in another study , goldberg et a1 . also noted that patients with glaucoma had significantly poor qol as compared to subjects without glaucoma . we noted that in patients with worse visual acuity and those who had to be started on more than two medications , qol further worsened after initiation of medical therapy . this in turn highlights the importance of educating the patients about the nature of the disease and ensuring drug compliance despite a perceived deterioration in symptoms after starting antiglaucoma medications . a comparison between poag and pacg subjects suggested a possible worse qol in the latter . a number of studies have elaborated on the significant problems encountered by subjects with glaucoma . reported that activities involving glare , dark adaptation , central and near vision , peripheral vision , and outdoor mobility were most problematic for patients with glaucoma . in another study , mckean - cowdin et al . noted that persons with visual field loss had the greatest difficulty with driving activities , dependency , mental health , distance vision , and peripheral vision wu et al . reported that the presence of open - angle glaucoma was significantly associated with lower scores for distance activities , mental health , and color and peripheral vision . showed that difficulty with bright lights , and with light and dark adaptation ; were the most frequently reported symptoms related to visual function in glaucoma patients . these findings are comparable to the results obtained in our study whereby glaucoma subjects were noted to have significant problems in activities which involved near and distance vision , night vision , ambulation , dark adaptation , color vision and glare . both worse eye and better eye visual acuity have been independently associated with poor qol . we noted that the decrease in better eye visual acuity was associated with worse qol at baseline and at 3 months . they also noted that qol had a significant correlation with better eye visual fields and visual acuity varied from 0.07 to 0.18 , however that the strength of correlation was weaker as compared to our results . van gestel et al . found a significant relationship between qol and iop in the better eye ( p < 0.001 ) . have published that both visual field index and visual acuity have a linear correlation with glaucoma - related qol . however , to assess the qol in patients with primary adult - onset glaucoma , all patients with secondary glaucoma were excluded . also , the exclusion of patients with cataract attempted to remove the confounding of the results obtained . second , the effects of economic factors , which are especially important for people residing in developing countries , were not included in the questionnaire . nonetheless , this study shows the important implication of presence of glaucoma , suggesting that newly diagnosed patients may have manifested symptoms of the disease , and initiation of medical therapy may be associated with further worsening of qol . thus , eye care providers should take every opportunity to educate patients about the nature of disease . they should be encouraged to continue with therapy despite no apparent benefits or even worsening of their qol at the outset .

objectives . to review the current protocols used for management of ludwig 's angina and to assess the efficacy of conservative measures in these cases . methods . a retrospective review of patients who were admitted to our institution for management of ludwig 's angina between 2003 and 2010 . results . two patients were identified . both were managed successfully with conservative measures and close airway observation . none needed an emergency intubation or surgical tracheostomy . there were no mortalities , and both had a short hospital stay . conclusion . recently , management of ludwig 's angina has evolved from aggressive airway management into a more conservative one . this is based on close airway observation on a specialised airway unit and a serial clinical airway assessment . improved imaging modalities , antibiotic therapy , surgical skills , and clinical experience are the key factors behind this change in practice . purpose : to evaluate the impact of initial topical medical therapy on newly diagnosed glaucoma patients using the indian vision function questionnaire ( ind - vfq33).patients and methods : the ind - vfq33 was used to evaluate the quality of life ( qol ) in 62 newly diagnosed patients with moderate to severe primary glaucoma and 60 healthy controls . ind - vfq33 is a 33 item qol assessment tool with three domains : general functioning , psychosocial impact and visual symptoms . the glaucoma patients were started on medical therapy and the qol assessment was repeated after 3 months.results:glaucoma patients ( mean age : 55.6 9.6 years , range 4077 years ) and controls ( mean age : 54.9 6.7 years , 4273 years ) were matched with respect to age ( p = 0.72 ) , gender ( p = 0.91 ) and literacy ( p = 0.18 ) . glaucoma patients had significantly worse qol as compared to controls at baseline across all the three domains ( p < 0.001 ) . 3 months after initiation of treatment , the overall qol life significantly worsened from baseline with a decrease in general functioning ( p < 0.001 ) and psychosocial impact ( p = 0.041 ) . visual acuity in better eye significantly co - related to poor qol at baseline ( p < 0.001 ) and at 3 months ( p = 0.04 ) . in addition , the use of > 2 topical medications significantly co - related to poor qol at 3 months ( p = 0.01).conclusions : evaluation using the ind - vfq33 revealed that newly diagnosed glaucoma patients have a significant worsening of qol after initiation of topical ocular hypotensive therapy . this should be an important consideration when educating patients about the disease and its therapy .

cyclosporine a has improved allograft survival and the quality of life for solid - organ transplant recipients . its effectiveness in transplantation by suppression of the immune system has led to its use in treating autoimmune diseases . csa inhibits the immune system by binding to cyclophilin , this complex then inhibits calcineurin , which in turn inhibits the translocation of the nuclear factor of activated t cells ( nfat ) and subsequent gene transcription . calcineurin inhibitors ( cni ) can cause nephrotoxicity involving acute renal vasoconstriction progressing on to glomerulosclerosis , tubulointerstitial fibrosis , and renal failure . due to cni - induced nephrotoxicity , the use of the immunosuppressive agent sirolimus ( srl ) in transplantations is becoming more widespread . srl has a different mechanism of immunosuppression compared to cnis , and a lesser degree of nephrotoxicity is observed with srl compared to that observed with the calcineurin inhibitors [ 4 , 5 ] . when administered with a cni , srl has been suggested to have a protective role when administered in conjunction with csa [ 6 , 7 ] . the rapamune us study group conducted a large multicentre clinical trial in which the efficacy of srl compared to azathioprine for reducing acute renal allograft rejection was investigated . the use of srl reduced occurrence and the severity of biopsy - proven acute rejection episodes . however , there have also been studies indicating enhanced nephrotoxicity when srl and csa are used in combination [ 9 , 10 ] . in another study , the authors showed that a combination of srl and csa significantly potentiated the nephrotoxic actions of csa by augmenting transforming growth factor ( tgf- ) . tgf- has been implicated as a major factor in the development of chronic cni toxicity . increased tgf- levels have been observed in renal cells exposed to csa , in animal models of csa toxicity and in patients with cni nephropathy [ 1215 ] ; however , the nephrotoxicity caused by csa remains to be fully elucidated . in this study , our hypothesis was to determine whether csa or srl had direct detrimental effects on the glomerulus and identify the possible mechanisms involved , using glomerular mesangial cells . human mesangial cells ( hmcs ) are key cells of the glomerulus and have an important role in regulating glomerular structure and function and have the potential to contribute to glomerulosclerosis by secreting profibrotic mediators , which can alter extracellular matrix ( ecm ) balance and disrupt renal function . this may be an important mechanism in renal disease progression and the upstream signalling pathways involved in csa - induced renal dysfunction are not well characterised and warrants further investigation . one intracellular pathway that may potentially be involved in immunosuppressive - induced renal damage is that of the mitogen - activated protein kinase ( mapk ) family , which has been implicated in tgf--induced cytotoxicity . the analysis of the differential activation of this pathway may provide a novel insight into the mechanisms of nephrotoxicity caused by csa . the hmcs were grown in rpmi 1640 containing 5% fcs , penicillin , streptomycin , and l - glutamine and maintained at 37c in a humidified atmosphere containing 95% air , 5% co2 . cells used in the inhibitor studies were pretreated for 1 hour with uo126 , prior to incubation with each drug treatment . mesangial cell viability was assessed using the resazurin conversion ( sigma - aldrich , 7017 ) cell viability assay . the viability of the cells was expressed as a percentage of the absorbance recorded for control cells . total rna was isolated using the trizol method from hmcs , according to the manufacturer 's protocol ( sigma - aldrich , t9424 ) . 1 g of total rna was used to synthesis cdna . a real - time pcr taqman assay was used to quantify the relative expression levels of genes of interest and has been described previously . briefly , cdna was amplified on the abi 7900ht sequence detection system at default thermal cycling conditions : 2 min at 50c , 10 min at 95c for enzyme activation , and then 40 cycles of 15 sec at 95c for denaturation and 1 min at 60c for annealing and extension . were used for all genes of interest , tgf- ( hs99999918_m1 ) , ctgf ( hs00170014_m1 ) , and mmp-9 ( hs00234579_m1 ) . the ribosomal 18s gene was used as an endogenous control for normalisation of the target genes . a negative control containing all reaction components except for superscript for each set of samples was used . a tgf-1 elisa was used to determine the effect csa had on secreted tgf-1 protein levels in the media of hmcs . the specificity and sensitivity of the assay was assessed using 5 ng of tgf-1 as a positive control and sterile water as a negative control . transfection reagent - dna complex was prepared by adding serum - free medium to a sterile eppendorf tube . fugene ( roche ) reagent was added directly to the tube and then 1 g of tgf- smad responsive ( caga ) luciferase reporter dna plasmid ( a gift from dr . roel goldschmeding ) and 1 g of control plasmid ( renilla ) ( promega ) . for multiple - well following transfection , cells were treated with each immunosuppressive agent for the required time period . cells were lysed using passive lysis buffer ( promega ) and put into fresh tubes . the luminometer was programmed to perform a 2-second premeasurement delay followed by a 10-second measurement . equal volumes of cell lysate and luciferase reagent were added together and the luminescence reading recorded . stop and glo reagent was added to halt the reaction and another reading performed . control lysates were made from untransfected cells to determine background luminescence levels and a positive control of cells treated with 5 ng tgf- was used . total protein was isolated from hmcs using the ripa buffer method ( sigma - alridch , r0278 ) according to the manufacturer 's protocol . expression levels of renal proteins following csa treatment was determined by western blot and has been described previously [ 15 , 20 , 21 ] . proteins of interest were detected using the following antibodies according to the manufacturer 's protocol ( rabbit anti - erk 1/2 , cell signalling technology , 9211s and 9211 ) . data was analysed by one - way analysis of variance ( anova ) , and multiple comparisons between control and treatment groups were made using the bonferroni posttest . the following scheme was used throughout the work ; * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . microscopic examination highlighted that treatment with csa or srl alone for 24 hours had no significant effect on hmc morphology ( figure 1(a ) ) . however , 24-hour treatment with csa / srl combination did cause alterations in hmc morphology ( figure 1(a ) ) . an elongation of normal hmc shape accompanied with gaps in the cell monolayer and cell death ( indicated by round floating cells ) was observed . treatment for 24 hour with csa or srl alone had no significant effect on hmc viability . treatment with csa / srl drastically reduced hmc viability ( 100 versus 54 3.9 , p < 0.05 ) . the concentrations of csa and srl used in this study were chosen following dose response studies , and it approximates to concentrations used in vivo and reflect concentrations in the kidney . treatment with csa for 24 hours caused a significant increase in tgf-1 secretion ( 843.1 7.1 versus 1089.5 26.1 pg / ml ; p < 0.05 ) as demonstrated by elisa ( figure 1(b ) ) . however , cotreatment of csa / srl caused the most significant rise in tgf-1 secretion ( 843.1 7.1 versus 1247.4 10.4 pg / ml ; p the increase in tgf-1 secretion observed with csa / srl was significantly greater than the increase observed with csa alone ( 1089.5 26.1 versus 1247.4 10.4 pg / ml ; p < 0.05 ) . to further examine the role of tgf- , we decided to investigate the effects of csa , srl , and csa / srl cotreatment on a smad - responsive luciferase construct . csa treatment caused a significant increase in smad activation ( 100 versus 156.9 1.7 luciferase units ; p < 0.05 ) . srl administration did not significantly alter smad activation ( 100 versus 123.5 4.3 luciferase units ) . treatment with csa / srl cotreatment caused the greatest increase in smad activation ( 100 versus 210.1 15.9 luciferase units ; p < 0.01 ) . the increase in smad activation in the presence of csa / srl cotreatment was significantly greater than with csa alone ( 156.9 1.7 versus 210.1 15.9 luciferase units ; p < 0.01 ) ( figure 1(c ) ) . the observation of increased smad signalling prompted an investigation into a downstream mediator of tgf- , connective tissue growth factor ( ctgf ) . treatment for 24 hours with csa showed a trend towards an increase in ctgf gene expression , which was not statistically significant . however , treatment with csa / srl cotreatment caused a significant increase in ctgf gene expression ( 1 versus 2.0 0.02 ; p < 0.01 ) ( figure 2(a ) ) . csa treatment for 24 hours caused a significant decrease in matrix metalloproteinase 9 ( mmp-9 ) gene expression ( 1.0 versus 0.18 0.15 ; p < 0.05 ) . csa / srl cotreatment also caused a significant decrease in mmp-9 gene expression ( 1 versus 0.37 0.2 ; p < 0.05 , figure 2(b ) ) . in order to determine the mechanism of csa / srl co - treatment - induced alterations , we investigated the effects of csa , srl , and csa / srl on erk 1/2 activation . csa significantly increased erk 1/2 phosphorylation after 15 , 30 , and 60 minutes administration ( figure 3(a ) ) . srl also significantly increased erk 1/2 phosphorylation after 15 and 30 and 60 minutes treatment ( figure 3(b ) ) . csa / srl co - treatment caused a synergistic increase in erk 1/2 phosphorylation at 15 , 30 , and 60 minutes . however , the combination of csa / srl co - treatment also significantly increased erk 1/2 phosphorylation at 24 and 48 hours , unlike the csa and srl individual treatments ( figure 3(c ) ) . the addition of uo126 prevented the csa , srl , and csa / srl co - treatment - induced stimulation of erk 1/2 ( figure 4 ) . total levels of erk 1/2 protein were unaltered following csa , srl , or csa / srl combination in the presence or absence of uo126 treatment . it was upon the notable activation of erk 1/2 phosphorylation that the effect of 24-hour csa / srl treatment in the presence of the erk 1/2 inhibitor uo126 was examined ( figure 5 ) . erk 1/2 inhibition appeared to partially ameliorate the csa / srl co - treatment - induced morphological alterations in hmcs . the csa / srl - induced reduction in hmc viability was prevented to some extent by erk inhibition ( 54 3.9 versus 88 3.5 , p < 0.001 ) . the addition of the erk inhibitor alone caused no effect on hmc viability or morphology . inhibition of erk 1/2 phosphorylation prevented the csa alone and csa / srl co - treatment - induced increases in tgf- secretion ( figure 4 ) . ( csa + uo126 , 518 23.5 versus 1089 26.1 , csa , p < 0.001 ) . ( csa / srl + uo126 , 424 54.2 versus 1247 10.4 , csa / srl , p < 0.001 ) . erk inhibition also dramatically reduced the csa / srl - induced increase in ctgf gene expression to below control levels ( csa / srl , 1.7 0.2 versus 0.6 0.14 , csa / srl + uo126 ) . erk inhibition also prevented the previously observed csa / srl co - treatment - induced decrease in mmp-9 gene expression ( 0.37 0.25 versus 1.6 0.2 ; p < 0.001 ) ( figure 4 ) . erk inhibition also restored the csa- and srl - induced decreases in mmp-9 and restored mmp-9 values to control levels . the results presented in this body of work demonstrated that the combination treatment of csa and srl did not result in an attenuation of the cni - associated changes in hmcs but in fact resulted in synergistic nephrotoxic - like effects . it appeared that the primary causative factor for the csa / srl - induced mesangial cell dysfunction was a significant dual increase in tgf- and ctgf expression . we also observed enhanced activation of the erk 1/2 mapk pathway , the tgf- signalling pathway , and alteration of ecm regulators . altered hmc morphology and viability gave the first indication that csa and srl combination treatment was exerting a detrimental effect when administered to hmcs in the present study . these initial findings of enhanced toxicity were surprising as it had been previously reported that srl had a protective role and could reduce the toxicity observed with cni administration [ 22 , 23 ] . however , more recently , experimental evidence has emerged suggesting that a combination of csa and srl might not be protective . in a rat model , kidney function and morphology were assessed following short - term combination therapy with csa , and srl . striped fibrosis and glomerular filtration rate ( gfr ) were significantly worse in rats receiving csa and srl combination according to the study by nielsen et al . . we observed that the secretion of tgf- was significantly increased following immunosuppressive drug treatment in the present model . tgf- is a major factor involved in csa - induced renal fibrosis and renal disease . tgf- has been shown to be upregulated in a number of in vitro and in vivo experimental models following csa treatment and in patients undergoing csa therapy [ 14 , 15 , 25 ] . increased tgf- may have contributed to the adverse effects observed in the present study . the lack of an increase in tgf- as observed with srl alone in the current study may help to explain the reduced nephrotoxic side effects associated with srl . it is accepted now that altered pharmacokinetics have a role in contributing to the observed toxicity with a combination of srl and csa . an in vitro study in human renal epithelial cells demonstrated that srl prolonged the intracellular accumulation of csa when given in combination . the authors showed that srl inhibited p - glycoprotein - mediated efflux contributing to csa nephrotoxicity . however , the reasons for the augmented nephrotoxicity resulting from a combination of srl and csa still remain to be fully defined , with some studies attributing the effect to pharmacokinetics and others to hyperglycaemia [ 10 , 26 , 27 ] . another known contributor to csa nephrotoxicity is tgf-. in the present hmc model , srl had a synergistic effect on tgf- secretion when coadministered with csa . however , similar increases in tgf- were also reported in a study on csa and srl combination therapy by shihab et al . . in that study , rats were given doses of csa alone , srl alone , or a combination of csa and srl . the authors observed a worsening in renal function in the group that were given csa and srl in combination compared to csa and srl alone . the csa / srl group also exhibited tubular injury , interstitial fibrosis , and arteriolopathy as indicated by histological analysis . elevated tgf- protein levels were also observed in the csa and srl co - treatment groups , and the levels were significantly higher than with csa or srl alone . to our knowledge , however , this is the first time that this effect has been observed in hmcs . ctgf was upregulated in the present hmc model following csa / srl co - treatment . ctgf has been found to be upregulated in many inflammatory glomerular diseases and in patient biopsies with numerous different conditions including glomerulonephritis and focal segmental glomerulosclerosis . it appeared that a novel dual elevation of tgf- and ctgf in the hmcs following csa / srl co - treatment contributed to the enhanced csa / srl - induced mesangial cell alterations . this dual elevation of tgf- and ctgf was not observed following treatment with csa or srl alone . in the current study , csa the observed reduction in mmp-9 in the present mesangial cell model may have facilitated ecm accumulation by preventing ecm degradation . it is known that ecm regulators are extremely context specific and chronologically variable . in models of chronic kidney disease , decreased mmp-9 has been shown to aid disease progression . in a rat ischemia - reperfusion model , caron et al . observed that mmp-9 was significantly upregulated and may have contributed to glomerular injury . in another model , transgenic mice overexpressing renin developed hypertension - induced renal dysfunction . isolated glomeruli from these mice exhibited elevated tgf- accompanied with decreased mmp-9 gene expression . these alterations in hmcs following csa / srl treatment are at least in part due to enhanced signalling through the erk 1/2 pathway . previous studies in our laboratory have indicated that the mapk pathways are also activated in response to csa in madin darby canine kidney ( mdck ) epithelial cells [ 32 , 33 ] . a biphasic activation of erk 1/2 following csa / srl co - treatment was observed in the hmcs and may be the mechanism by which the synergistic nephrotoxic effect is exerted by the csa / srl co - treatment . this is further supported by the fact that erk 1/2 inhibition resulted in significant attenuation of this csa / srl - induced hmc dysfunction . considering the well - characterised roles of erk 1/2 signalling in cell survival and proliferation , our finding that enhanced erk 1/2 signalling is central to csa / srl cytotoxicity is interesting . this apoptosis was accompanied by activation of the erk 1/2 mapk pathway . pharmacological inhibition of erk 1/2 similarly , in cisplatin - induced cytotoxicity , erk 1/2 has been shown to play a central role . several mechanisms of erk 1/2-mediated cytotoxicity have been proposed including erk - dependent activation of caspases and induction of autophagy . importantly from the perspective of this study , in examples of erk - mediated cytotoxicity , erk activation is unusually prolonged ( up to 72 hours ) . therefore , the prolonged erk activation observed in this study , in the presence of csa / srl co - treatment , may be a key mechanistic difference compared to the two immunosuppressants in isolation . this suggests that the erk 1/2 pathway may play a major role in tgf- signalling . erk 1/2 also appeared to play a role in ctgf expression in this mesangial cell model as ctgf gene expression was attenuated by erk inhibition . these observations have been observed in other models of csa nephrotoxicity [ 39 , 40 ] . our proposed mechanism for the enhanced csa / srl nephrotoxicity is that csa / srl strongly activates erk 1/2 , which promotes tgf- cellular secretion . tgf- may also promote decreased ecm degradation via decreased mmp9 , which contributes to the observed mesangial cell dysfunction . these findings are important in determining future immunosuppression strategies for srl therapy in clinical organ transplantation . currently , there are two main strategies : de novo use of srl in combination with reduced amounts of csa or complete conversion from a cni - based protocol to srl in cases where well - recognized adverse effects of cnis ( such as impaired renal function ) are prevalent . however , there is much debate on this issue and longer - term studies in larger cohorts of patients are required to determine whether replacement of csa with srl provides any significant improvement in patient and graft survival . the results shown in this study would suggest that long - term use of srl in patients will have serious nephrotoxic effects . this work was supported by the health research board , science foundation ireland , enterprise ireland , and the programme for research in third level institutions administered by the higher education authority and by the eu 7th framework grant syskid , health - f2 - 2009 - 241544 . c. slattery is funded by a government of ireland research fellowship from the irish research council for science , engineering and technology . the results presented in this paper have not been published previously in whole or part , except in abstract form . to identify the most probable asian parental groups of the malagasy , we adopted a two - stage approach : first , we identified the most likely proxy populations using a data set with wide geographical coverage but relatively low density of single nucleotide polymorphism ( snp ) , then followed by a higher density snp data set that gives increased statistical power . this allows us to reconstruct the admixture processes that led to the emergence of modern malagasy . the admixture profile of our data set ( 2,183 individuals from 61 populations genotyped for 40,272 snps ; supplementary figs . s2 and s3 , 2009 ) , shows that the malagasy genetic diversity is best described as a mixture of 68% african genomic components and 32% asian components , corresponding well with the results of previous studies ( capredon et al . while the african ancestry component in malagasy appears to be broadly similar to that still present today in south african bantu , the asian ancestry presents a more complex pattern . this complexity is the key reason why previous studies have been unable to point firmly to a unique asian source , making any subsequent anthropological inferences debatable ( pierron et al . this problem arose with a study of the maanyan population of borneo , whose language has long been identified as the closest to the malagasy language ( dahl 1951 ) , but who surprisingly exhibit no clear genetic connection to malagasy ( kusuma et al . , the higher genetic complexity of the asian ancestry component in malagasy likely reflects the fact that more than a single source population was involved in its formation . affinities to the malagasy asian components are found at high frequency across several island southeast asian groups , but notably in malay , a dominant group of ancient seafaring traders ( first millennium ce ) , and admixed groups from borneo ( i.e. , banjar , ngaju , south kalimantan dayak , lebbo , murut , dusun , and bidayuh ; supplementary fig . the connection between malagasy , and the borneo and malay populations , is supported by f3-statistics ( z - scores<2 ; supplementary table s2 , supplementary material online ) ( patterson et al . 2012 ) and treemix analyses ( 35% of malay / borneo gene flow to malagasy ; supplementary fig . however , to more specifically identify the asian ancestry of the malagasy genome , we performed a local ancestry analysis with pcadmix ( brisbin et al . 2012 ) using two proxy parental meta - populations comprising 100 individuals with african ancestry ( randomly selected from yoruba , south african bantu , kenyan luhya , and somali groups ) and asian ancestry ( randomly selected from chinese , philippine igorot , bornean maanyan , and malay groups ) . masking the haplotypes inferred to derive from africa , we performed an ancestry - specific principal components analysis ( pca ) ( patterson et al . both show that the asian genomic components of malagasy cluster tightly with southeast borneo groups ( banjar , south kalimantan dayak , ngaju , and maanyan ) ( 1,664 snps ; fig . this connection is supported by the highest f 3-statistics and the lowest fst genetic distances also being observed between asian ancestry of the malagasy and these same southeast borneo groups ( f3 > 0.12 ; fst < 0.02 ; fig . 1b ; supplementary tables s3 and s4 , supplementary material online ) . fig . 1localization of the asian ancestry of malagasy by ( a ) a treemix dendrogram , ( b ) fst distances and f3 statistics , all based on the asian - snp data set , and ( c ) a shared identity - by - descent ( ibd ) analysis based on haplotypes inferred from the high density of snp data set . ( a ) the treemix dendrogram was inferred imposing no a priori assumptions of migration , displaying only the tree topology . ( b ) values of the f3 ( asian - snp vezo , x ; yoruba ) statistics are represented by dots with standard error bars . the color of each dot corresponds to the fst distances between the asian ancestry of the vezo and each asian population using a gray - yellow - red color scale from the highest ( 0.196 ) to the lowest values ( 0.02 ) . ( c ) the cumulative shared ibd ( mb ) between pairs of malagasy : asian individuals were averaged to obtain one value of ibd sharing per asian population . the numbers 115 correspond to the populations presented on the treemix dendrogram with the addition of 16 which stands for brahmin indian . localization of the asian ancestry of malagasy by ( a ) a treemix dendrogram , ( b ) fst distances and f3 statistics , all based on the asian - snp data set , and ( c ) a shared identity - by - descent ( ibd ) analysis based on haplotypes inferred from the high density of snp data set . ( a ) the treemix dendrogram was inferred imposing no a priori assumptions of migration , displaying only the tree topology . ( b ) values of the f3 ( asian - snp vezo , x ; yoruba ) statistics are represented by dots with standard error bars . the color of each dot corresponds to the fst distances between the asian ancestry of the vezo and each asian population using a gray - yellow - red color scale from the highest ( 0.196 ) to the lowest values ( 0.02 ) . ( c ) the cumulative shared ibd ( mb ) between pairs of malagasy : asian individuals were averaged to obtain one value of ibd sharing per asian population . the numbers 115 correspond to the populations presented on the treemix dendrogram with the addition of 16 which stands for brahmin indian . to explore this connection in more detail , we turned to the high density snp data set ( 551 individuals from 24 populations genotyped for 374,189 snps ; supplementary table s1 , supplementary material online ) . we confirmed the earlier result that malagasy have the highest values of cumulative shared identity - by - descent fragments with southeast borneo populations ( fig . s6 , supplementary material online ) . to expand on this , however , we inferred the population sources of the malagasy , their relative ratios and the dates of potential admixture events with globetrotter ( hellenthal et al . 2014 ) , defining each population in our data set as a donor / surrogate group and the malagasy as the recipient , using the haplotype painting data obtained with chromopainter ( lawson et al . the best fit outcome for the malagasy was obtained under a model of a single admixture event between two sources : the banjar representing 37% of modern malagasy and the south african bantu population representing the other 63% ( r=0.99 , p < 0.01 ; fig . 2 and supplementary table s5 , supplementary material online ) . the admixture event was dated to 675 years bp ( 95% ci : 625725 years bp , supplementary table s5 , supplementary material online ) , which is similar to the dates of admixture estimated by alder ( 550750 years bp ) using banjar population in combination with the south african bantu ( supplementary table s6 , supplementary material online ) ( loh et al . when each malagasy ethnic group is analyzed separately , similar parental populations , admixture proportions , and dates are obtained with the noticeable older estimated dates toward the east coast of madagascar ( supplementary table s5 , supplementary material online ) . 2scenario for the asian genetic ancestry in malagasy based on the best fit models inferred by globetrotter . the brown circle represents the malagasy ( bottom left ) , while the green circle represents the banjar ( right ) . red semicircles show the african ancestry ( south african bantu ) , whereas the other semicircles represent asian ancestry from malay ( blue ) , banjar ( green ) , and maanyan ( yellow ) . the arrows show migration events , with indicative routes , inferred in our analyses with dates of admixture in italic estimated by globetrotter . dates in bold correspond to dates of migration estimated from archaeological and historical data ( beaujard 2012a ) . scenario for the asian genetic ancestry in malagasy based on the best fit models inferred by globetrotter . the brown circle represents the malagasy ( bottom left ) , while the green circle represents the banjar ( right ) . red semicircles show the african ancestry ( south african bantu ) , whereas the other semicircles represent asian ancestry from malay ( blue ) , banjar ( green ) , and maanyan ( yellow ) . the arrows show migration events , with indicative routes , inferred in our analyses with dates of admixture in italic estimated by globetrotter . dates in bold correspond to dates of migration estimated from archaeological and historical data ( beaujard 2012a ) . crucially , these dates of genetic admixture , in agreement with a previous study ( pierron et al . 2014 ) , reflect the midpoint or end of noticeable admixture between groups of asian and african ancestry in madagascar , rather than the start of this contact . therefore , they could correspond to the end of the period of the main austronesian presence in madagascar that started around the first millennium ce ( dahl 1951 , 1991 ; dewar and wright 1993 ; adelaar 1995 ; cox et al . 2012 ; adelaar forthcoming ) . on the other hand , around 1100700 years bp , climatic changes in the south of africa forced bantu populations to move to more hospitable places ( huffman 2000 ) . this south bantu migration has previously been suggested as an explanation for the higher density of populations observed in the south of madagascar ( beaujard 2012a ) . as all of our sampled groups live in the south of madagascar , and considering that the estimated dates of admixture are more recent on the west coast ( supplementary tables s5 and s6 , supplementary material online ) , it is tempting to interpret our admixture date as marking the last significant bantu migration to madagascar , perhaps initiated by climatic changes in africa . these analyses clearly identify an austronesian - speaking population , the banjar in the southeast region of borneo , as the closest asian sources for modern malagasy . linguistic reconstruction of the proto - malagasy language indicated that it appears to be derived mainly from the southeast barito language spoken today by the maanyan ( dahl 1951 ) , a southeast borneo group . however , we have previously shown ( kusuma et al . 2016 ) , and reconfirm here , that in genetic analyses , the maanyan are only distantly related to the malagasy in terms of genetics . interestingly , the linguistic studies indicated a noticeable proportion of malay words in present - day malagasy languages ( adelaar 1989 , 2009b ) . we estimated the best fit scenario for the admixture process by modeling the current banjar diversity with globetrotter ( hellenthal et al . 2014 ) . the genetic diversity of the banjar best fits a model of a unique admixture event ( r=0.62 ; p < 0.01 ) between two major ancestries that can be traced back to malay ( 77% ) and maanyan ( 23% ) ( fig . 2 and supplementary table s5 , supplementary material online ) . we estimate the date of the last noticeable admixture approximately 425 years bp ( 95% ci : 275500 years bp ) . since the banjar originated from a maanyan - malay admixture , at a time preceding the supposed date of migration to madagascar ( i.e. , 1000 years bp ) , the ancestors of the banjar were presumably speaking a language close to that reconstructed for proto - malagasy ( adelaar forthcoming ) . although we can not fully exclude that the malay - maanyan admixture occurred in malagasy , prior to the bantu gene flow , the observed haplotypic structure is so similar to the ones observed in the banjar that it is more parsimonious to interpret this admixture to have happened first in borneo . this analysis reconciles both the linguistic and genetic data , strengthening our scenario placing the banjar as the main asian parental populations of the malagasy . their current genetic diversity appears to be the reflection of the historical relationship between madagascar , southeast borneo , and the malay . the maritime routes linking madagascar to borneo were particularly exploited during the rapid expansion of trading networks led by the hindu malay kingdoms , such as srivijaya ( 6th13th centuries ) ( ras 1968 ; beaujard 2012a ) . established on the islands of sumatra and java , the malay traded with far - distant regions , notably across east asia and reaching as far as east africa ( beaujard 2012a ) . their influence increased across all the southeast asian islands , notably in borneo where they established several trading posts , such as one in the city of banjarmasin in southeast borneo ( ras 1968 ; beaujard 2012a ) . as related in the only banjarese historical records available , the hikayat banjar ( tale of banjar ) ( ras 1968 ) , the main city of the banjar population was a major trading post in the former malay empires . this probably favored interactions with inland groups in borneo , such as the maanyan , but also with other populations such as the bajo sea nomads ( supplementary fig . s6 and table s5 , supplementary material online ) ( adelaar 2009a ; beaujard 2012a ; kusuma et al . the malay domination of trading networks collapsed during the 15th16th centuries , with the emergence of several sultanates and the arrival of europeans , which could correspond to the end of noticeable malay gene flow into the banjar population , as indicated by our estimated date of admixture between malay and maanyan around 425 years bp . our study provides strong support for a new scenario for the austronesian settlement of madagascar , reconciling cultural , linguistic , and genetic data , in which the banjar population played key roles in establishing the asian founder population of the malagasy . the malay trading networks during the first millennium ce triggered one of the earliest protoglobalization processes , bringing southeast asian populations to east africa ( beaujard 2012a ) . the banjar , currently living in southeast borneo , show the highest affinity to the main genetic ancestry component in malagasy and can therefore be suggested to be likely to have been the ethnic group that accompanied the malay in their maritime voyages to madagascar . this population with composite ethnic ancestry emerged from the long - standing presence of malay in borneo , creating an admixed community with local austronesian - speaking groups , such as the maanyan . before the probable date of migration ( around 1000 years bp ) , the ancestors of the current banjar would have contained both malay and maanyan genetic diversity , and probably linguistic inheritances from both ( adelaar forthcoming ) . although the exact maritime route(s ) of migration from borneo to madagascar are still an open question , our study identifies the flow of malay - maanyan genomic ancestries as carried by banjar ancestors as the source to malagasy . all experimental and analytical procedures are described in the supplementary method file , supplementary material online . this study was approved by the research ethics commission of the eijkman institute for molecular biology ( jakarta , indonesia ) .

end stage renal disease ( esrd ) is an ever increasing problem worldwide . however the mechanisms underlying disease progression are not fully elucidated . this work addressed nephrotoxicity induced by the immunosuppressive agents ' cyclosporine a ( csa ) and sirolimus ( srl ) . nephrotoxicity is the major limiting factor in long term use of csa . srl causes less nephrotoxicity than csa . therefore investigations into the differential effects of these agents may identify potential mechanisms of nephrotoxicity and means to prevent esrd induced by therapeutic drugs . using elisa , western blotting , quantitative pcr and a reporter gene assay we detailed the differential effects of csa and srl in human renal mesangial cells . csa treatment increased profibrotic tgf-1 secretion in human mesangial cells whereas srl did not , indicating a role for tgf- in csa toxicity . however we observed a synergistic nephrotoxic effect when csa and srl were co - administered . these synergistic alterations may have been due to an increase in ctgf which was not evident when the immunosuppressive drugs were used alone . the csa / srl combination therapy significantly enhanced smad signalling and altered the extracellular matrix regulator matrix metalloproteinase 9 ( mmp-9 ) . inhibition of the erk 1/2 pathway , attenuated these csa / srl induced alterations indicating a potentially significant role for this pathway . malagasy genetic diversity results from an exceptional protoglobalization process that took place over a thousand years ago across the indian ocean . previous efforts to locate the asian origin of malagasy highlighted borneo broadly as a potential source , but so far no firm source populations were identified . here , we have generated genome - wide data from two southeast borneo populations , the banjar and the ngaju , together with published data from populations across the indian ocean region . we find strong support for an origin of the asian ancestry of malagasy among the banjar . this group emerged from the long - standing presence of a malay empire trading post in southeast borneo , which favored admixture between the malay and an autochthonous borneo group , the maanyan . reconciling genetic , historical , and linguistic data , we show that the banjar , in malay - led voyages , were the most probable asian source among the analyzed groups in the founding of the malagasy gene pool .

in the urban areas of developing countries such as turkey , biomass ( eg , wood , animal dung , crop residues ) is used as a source of energy.1,2 exposure to biomass smoke is the leading cause of copd , especially in developing countries.3 a half of copd deaths are due to biomass exposure , and 75% of these deaths occur in females.3,4 females are more exposed to biomass smoke than men in turkey because of traditional lifestyles in rural areas . carbon monoxide , nitrous oxide , sulfur oxide , formaldehyde , and polycyclic organic matter , including carcinogens such as benzopyrene , are all products of biomass smoke.5 these microparticulate products reach the peripheral airways and produce adverse effects on pulmonary functions.6 the primary aim of this study was to determine the difference of pulmonary functions between females who were exposed to biomass smoke and those who were not and the secondary aim was to determine the time course relationship with altered pulmonary function test ( pft ) results and define the importance of intensity of smoke on pulmonary functions , with the calculation of a new index ( cumulative exposure index [ cei ] ) , which has not been described in the literature before . there is a tradition in the village of kaizman ( a borough of kars located in the eastern part of turkey ) that females are responsible for the cooking of bread in their houses . people are exposed to heavy smoke due to the cooking conditions ( lack of chimney and poor ventilation ; figure 1 ) . girls start making bread at the age of 15 years , and the daily exposure to smoke is between 2 hours and 4 hours ; they usually cook 1 day / wk , ie , two to four times per month . they use organic wastes for burning fire , eg , dry animal dung ( manure ) , rather than wood or charcoal , as the fuel source ( figure 2 ) . our only inclusion criterion was screening the pulmonary functions in females who were exposed to biomass smoke . we had visits to the village and were able to screen 474 females who were exposed to biomass smoke . smoking is the leading cause of copd ; therefore , we eliminated females who were current or former smokers . cardiovascular diseases are included in the comorbidities of copd , and a volume overload due to heart failure or renal failure may affect pft results negatively . asthma is another important disease that results in altered pft results ; therefore , females who had history of allergy and asthma were excluded . due to its inflammatory process , respiratory tract infection may narrow the airways with secretions and may affect pft results , and hence those who received therapy due to upper or lower respiratory infections in the last 1 month were excluded . the final study group comprised 115 females who were purely exposed to biomass smoke ( table 1 ) . a control group was generated with 73 healthy individuals who lived in the same area and had never been exposed to biomass smoke . all the research procedures were approved by the local ethical committee of the public health directorship in kars , and all participants provided written consent for the study . a pft device ( spirolab iii ; mir , waukesha , wi , usa ) was used on each patient after being calibrated by the technician , and forced expiratory volume in 1 second ( fev1 ) , forced vital capacity ( fvc ) , and forced expiratory flow during the 25%75% portion of the fvc ( fef 2575 ) were recorded for both the study group and control group . pft results were grouped in accordance with the guidelines of the european respiratory society and american thoracic society.7 fef 2575 < 60% was accepted as small airway , fev1/fvc < 70% was accepted as obstructive , and fev1/fvc > 70% plus fvc < 80% was accepted as restrictive lung disease . after pfts were performed in the study group and control group , the differences between the pft results were evaluated . in addition , the age at which the participants started to cook and hours per day , weeks per month , and total years of cooking were questioned . these variables correlated with the pft results in the study group . the risk of duration in years for existence of pathologic pft results was calculated . in addition , we tried to formulate an index ( cei ) like cigarette pack year to provide data for intensity of biomass exposure . this index was defined by multiplying the duration of biomass exposure ( in years ) with the frequency of exposure ( hours per day , weeks per month ) and dividing the result by three . finally , the correlation between cei and pathologic pft results was defined . cei = hours per dayweeks per monthexposure duration in year3(1 ) statistical package for the social sciences 21.0 ( ibm corporation , armonk , ny , usa ) and medcalc statistical software ( medcalc software , ostend , belgium ) were used for statistical analysis . descriptive statistics ( frequency , percentage , mean [ min max ] ) were used to evaluate the study data . the independent sample t - test was used to compare parameters between the groups . a one - way analysis of variance test was used to compare the parameters between more than one group , and the bonferroni test was used to determine the group that showed significant difference . pearson s correlation analysis was used to evaluate the relationship between pft and independent variables ( age , body mass index , age of starting cooking and hours per day , weeks per month , and years spent cooking ) . in addition , linear regression analyses were performed to evaluate the correlation between independent variables and pft results . a receiver operating characteristic ( roc ) curve analysis was performed to determine cutoff levels for duration in years and cei for the existence of altered pft results ( small airway disease , obstruction , and restriction ) ; moreover , sensitivity and specificity were calculated based on the cutoff value . statistical package for the social sciences 21.0 ( ibm corporation , armonk , ny , usa ) and medcalc statistical software ( medcalc software , ostend , belgium ) were used for statistical analysis . descriptive statistics ( frequency , percentage , mean [ min max ] ) were used to evaluate the study data . the independent sample t - test was used to compare parameters between the groups . a one - way analysis of variance test was used to compare the parameters between more than one group , and the bonferroni test was used to determine the group that showed significant difference . pearson s correlation analysis was used to evaluate the relationship between pft and independent variables ( age , body mass index , age of starting cooking and hours per day , weeks per month , and years spent cooking ) . in addition , linear regression analyses were performed to evaluate the correlation between independent variables and pft results . a receiver operating characteristic ( roc ) curve analysis was performed to determine cutoff levels for duration in years and cei for the existence of altered pft results ( small airway disease , obstruction , and restriction ) ; moreover , sensitivity and specificity were calculated based on the cutoff value . a total of 188 females were evaluated in this study , of whom 115 had been exposed to biomass smoke and 73 had had no exposure to biomass smoke . the females who had been exposed to smoke were older than those in the control group , 33.47 years and 32.68 years , respectively . the study group and controls were grouped according to their pft results : small airway disease , obstruction , and restriction . the median fef 2575 value was 70.0% in the study group and 77.0% in controls ( p<0.001 ) . the median fev1/fvc value was 79.0% in the study group and 84.0% in controls ( p<0.001 ) . the median fev1 value was 78.0% in the study group and 80% in controls ( p=0.024 ) , and the median fvc value was 81.0% in the study group and 84.0% in controls ( p=0.036 ; table 2 and figure 3 ) . twenty - seven ( 23.8% ) females in the study group and four ( 5.5% ) in the control group had small airway disease . females who were exposed to biomass smoke had a higher probability of having small airway disease than those who were not ( =3.895 , p=0.038 ) . twenty - two ( 19.1% ) females in the study group and ten ( 13.7% ) in the control group had obstruction ( fev1/fvc < 70% ) . although the probability of having obstruction was higher in females who were exposed to biomass smoke , it was not statistically significant ( =0.933 , p=0.223 ) . twenty ( 17.3% ) females who were exposed to biomass smoke had restriction compared with ten ( 13% ) in the control group ( =0.982 , p=0.189 ; table 3 ) . we also evaluated correlations between pft results and some parameters such as age at starting to cook , how many hours per day and weeks per month spent cooking , and the duration of biomass exposure . the median values of the parameters for the patient group are shown in table 4 . in the study group , 27 ( 23.8% ) females had small airway disease and 22 ( 19% ) had obstructive airway disease . starting to make bread at younger age and more weeks and more years spent cooking were risk factors for the development of both small airway disease and obstructive airway disease , whereas hours per day spent cooking was not a risk factor ( table 5 ) . in the study group , when the parameters were evaluated in this group , it was observed that starting to cook at younger age , more hours per day , more weeks per month , and more years spent cooking were all risk factors for the development of restrictive lung disease ( table 5 ) . we also performed a correlation analysis between dependent ( fev1 , fev1/fvc , fvc ) and independent variables ( age at starting to cook , hours per day , weeks per month , duration in years ) . for fev1/fvc , the correlation analysis was statistically significant for duration of exposure to biomass smoke ; as the duration of biomass exposure increased , fev1/fvc was negatively affected . for fev1/fvc , conversely , with the exception of age of starting to cook , a negative correlation with all the independent variables for fvc was found ( table 6 ) . by using the roc curve analysis , we defined the duration for existence of small airway disease , obstruction , and restriction . the cutoff values were 16 years for small airway disease ( sensitivity : 100% , specificity : 76.9% ) , 17 years for obstructive lung disease ( sensitivity : 95.5% , specificity : 69.9% ) , and 17 years for restrictive lung disease ( sensitivity : 100% , specificity : 74.3% ; figure 4 ) . females who had been exposed to biomass smoke and had altered pft results had higher values of cei compared with females who had pft results with normal values ( table 7 and figure 5 ) . in addition , we calculated a cutoff value for pathologic pft results using the roc curve analysis . cei was 34.7 for the existence of small airway disease , 42.7 for obstruction , and 45.3 for restriction ( table 8 and figure 6 ) . this case control study has two major findings : first , females who are exposed to biomass smoke are at risk of developing altered pulmonary function ( both obstructive and restrictive lung disease ) , and second , the duration of exposure to biomass smoke is important for the probability of altered pulmonary function . in our study , both obstructive and restrictive lung diseases were observed among females who had been exposed to biomass smoke . chronic exposure to smoke may cause inflammatory reactions in the alveolar space and the bronchial wall.8 as expected , this was the reason for obstructive lung disease in our patients . in our study , we observed that as biomass exposure increased in quantity and intensity , the risk of appearance of copd increased . similar results were reported in the studies by ramrez - venegas et al9 and regalado et al.10 these authors reported that advanced stages of copd exist as the duration of biomass smoke increases . according to our data , females who are exposed to biomass smoke for > 17 years are at risk of developing copd . in the current literature , the risk of developing obstructive airway disease usually requires > 20 years of exposure.911 the females in our study were exposed to more concentrated smoke because of the poor conditions in the places in which they cooked ( figure 1 , no chimney and poor ventilation ) ; massive smoke exposure may be the reason for altered pft results of shorter duration . in addition to obstruction , we also observed restrictive pft results ( fev1/fvc > 70 , fvc < 80% ) in 22 ( 19.1% ) females in the study group . in the current literature , a few studies mention a restrictive pattern in biomass exposure : kara et al12 and arslan et al13 defined restrictive images ( opacities , fibrotic bands ) in computed tomography ( ct ) scans of patients who were exposed to biomass smoke . septal enlargements , goblet cell metaplasia , glandular hyperplasia , and fibrosis can be observed histopathologically when the source of energy is something other than wood.14 in our study , the restrictive results can be explained by the source of energy used by the females ( females - burned animal waste [ manure ] ; figure 2 ) . burning organic material such as manure may cause some kind of chemical reactions , which may be responsible for restrictive lung disease . although some studies have defined a correlation between restrictive lung disease and biomass exposure , none have mentioned the duration of exposure for the risk of developing restrictive lung disease.12,13 according to our data , females who are exposed to biomass smoke for > 17 years are at risk of developing restrictive lung disease . twenty - seven ( 23.8% ) females had small airway disease . in our study , small airway disease was observed among females who started to cook at younger ages , and the duration of exposure for the risk of developing small airway disease was 16 years . they claimed that exposure to biomass smoke at younger ages was not a risk factor for small airway disease . particles < 10 m in diameter , particularly those < 2.5 m , can penetrate deep into the lungs , especially into the peripheral airways.5,8 with the cei calculation , we tried to define the importance of the intensity of biomass smoke . in this study , we did not accept exposure to biomass smoke to be like cigarette packet years . cigarette smokers smoke continuously , but the people in our study were exposed to biomass smoke for only a few hours per day , one day per week , or nearly every week . prez - padilla et al16 and ekici at al17 described a calculation by multiplying hours per day and total years ; they defined that higher values from this calculation provided for greater probability of altered pft results . the main difference of their study from ours was that the number of days per week that people were exposed to biomass smoke was not added to their calculation . if it were possible to perform pulmonary imaging studies , particularly ct of the lungs , it would be possible to differentiate chronic bronchitis from emphysema and show reticulonodular appearance and fibrosis that could be suggestive for restrictive lung disease . if it were possible to perform pulmonary imaging studies , particularly ct of the lungs , it would be possible to differentiate chronic bronchitis from emphysema and show reticulonodular appearance and fibrosis that could be suggestive for restrictive lung disease . biomass exposure is a very important public health problem among females , especially in developing countries . the risk of developing problems in lung functions begins after exposure to smoke for > 15 years . precautions must be taken for populations living in these kinds of rural areas , and people must be educated about the adverse effects of biomass smoke . hepatitis b virus infection ( hbv ) is an inflammatory disease of liver due to double stranded virus of the hepadnaviridae family . hepatitis b infection possesses a major health concern and is the most common blood borne viral infection , placing health care workers and medical and dental professionals at higher occupational risk . the other mostly common communicable diseases include human immunodeficiency virus ( hiv ) and hepatitis c virus ( hcv ) . the possible forms of transmission of hepatitis virus include unprotected sexual contact , blood transfusion , reuse of contaminated needles , and vertical transmission from mother to child during pregnancy . in dental setting the most common mode of transmission is from percutaneous exposure ( needle stick injuries ) and also from contact with blood or saliva of infected patients . the possibility of hbv transmission from exposure to saliva and gingival crevicular fluid has been confirmed , which makes the oral health care professionals more vulnerable for hepatitis infection . nearly two billion people in the world have been infected by hbv and there are nearly 350 million people who are chronic carriers . hepatitis b and hepatitis c infection can become persistent and show the way to cirrhosis of liver and even liver cancer . it is mainly acquired in the course of contaminated needles or tainted blood products and infection patterns are diffuse . among the professionals , dentists are placed in high risk group as actual sufferers and carriers with a grim picture . it is of prime importance for all dental schools , medical staff , and dental staff to conduct talks and create awareness about hepatitis b infection . india has the intermediate endemicity of hepatitis b virus with surface antigen ( hbsag ) prevalence between 2% and 10% among the population studied . in india about four percent of the population was estimated to be hbv carriers giving a total pool of approximately 36 million carriers . chronic infection with hepatitis b may be either asymptomatic or may be associated with a chronic inflammation of the liver ( chronic hepatitis ) , leading to a cirrhosis over a period of several years . studies have shown that the risk of exposure for general dentists is about three to four times greater and for nonimmunized surgical specialists about six times greater than that of the general population . in the dental setting , there are special circumstances and opportunities which can lead to the transmission of such organisms to dental healthcare professionals and to dental clinical students . the incidence of hbv can be reduced by giving proper education regarding its transmission and immunizations to the public , all healthcare workers ( hcv ) , and students . there are no adequate data on the awareness of hepatitis among dental college students in india . hence this forms the base of the present study which aimed to analyze the awareness of hepatitis b infection among the clinical students in a private university . this is an institutional based study conducted to determine the awareness on hepatitis b infection among dental students in university dental college . this study is conducted among the dental students who are attending the third year , final year , and internship ( fifth year trainee ) of graduate program in saveetha dental college and hospital . the students who were present at the day of the particular study and were willing to participate were included in the study and those who were not willing to participate were not included in the study . all the third year , final year , and intern students who were present at the day of study were considered as the sample size for the study . this is a self - reported questionnaire which takes about 10 mins for completion . this included questions about the awareness on hepatitis b infection and the preventive measures taken by the dentist to protect both patients and dentists from hepatitis infection . all data were entered in a data entry form which was transferred to spss software ( spss , version 17 for windows ) . analysis of variance ( anova ) was performed for each variable to assess whether significant differences were observed between the three years of study and also within groups . a total of 150 students participated in the study . of these 34 ( 22.7% ) students were male and 116 ( 77.3% ) were female . the question regarding possible transmission of hbv through contact with saliva , only 23 ( 25.3% ) of the third year students were aware of the fact while 45 ( 30% ) of the final year students and 41 ( 27.3% ) of the interns were already aware of salivary transmission which showed a significant difference ( p = 0.000 ) . in response to the question regarding confirmed risk of hbv transmission through dental treatments 30 ( 20% ) of the third year students , 35 ( 23.3% ) of the final year students , and 32 ( 21.3% ) of the interns were aware of the possible risk during the procedure . regarding high risk of hbv infection for dentist 24 ( 16% ) of the third year students , 40 ( 26.7% ) of the final year students , and 41 ( 27.3% ) of the interns were aware of the high risk , which showed statistically significant difference between various academic years ( p < 0.05 ) . in response to the question on needle stick injuries 30 ( 20% ) of the third year students , 34 ( 22.75% ) of the final year students , and 35 ( 15% ) of the interns reported having such unintentional procedures . this variable did not show a statistically significant difference between various academic years ( p = 0.541 ) . questions regarding high risk of transmission of hbv than hiv through needle stick injury 26 ( 17.3% ) of the third year students , 31 ( 20.7% ) of the final year students , 25 ( 16.7% ) of the interns replied that risk for hiv transmission is more . twenty three 23 ( 15.3% ) of the third year students , 27 ( 18% ) of the final year students , and 36 ( 24% ) of the interns were aware that hbv transmission from dentist to patient can be prevented with the use of gloves which did not show a statistically significant difference between academic years ( p = 0.026 ) . to question regarding the level of knowledge about universal precaution 6 ( 4% ) of the third year students , 5 ( 3.3% ) of the final year students , and 6 ( 4% ) of the interns were not aware about the precautions . five ( 3.3% ) of the third year students , 14 ( 9.3% ) of the final year students , and 19 ( 12.7% ) of the internship students have heard something about universal precautions . nineteen ( 12.7% ) of the third year students , 27 ( 18% ) of the final year students , and 21 ( 14% ) of the interns were being educated about universal precaution . twenty ( 13.3% ) of the third year students , 4 ( 2.7% ) of the final year students , and 4 ( 2.7% ) of the interns were following an executable protocol for universal precaution . this variable shows a significant difference between the academic years ( p = 0.002 ) . table 2 shows the comparison between and within groups on the response to each question . the question regarding salivary transmission of hbv showed a significant difference among the students of various years . the question regarding higher predominance of dentist among the population for hbv infection also showed a significant difference ( p < 0.05 ) . regarding the universal precaution there was a significant difference between the various year students ( p < 0.05 ) . the overall level of knowledge about viral hepatitis among the various years is fairly satisfactory . the level of knowledge regarding hbv was fairly good among final and the interns when compared to 3rd year students . there is no formal school based health education for students in india , and this may also be considered as one of the important reasons for lower knowledge of hepatitis b among 3rd year students . the dental curriculum in india is such that students enter the clinical practice only in third year of study . it is because of this the study population included students only in the clinical year of study . to our knowledge , this is the first study investigating this topic among dental students in chennai , india . based on the results of this study , we can infer that there is need for improving the knowledge of hbv infection among the nonclinical students . when a student enters the final year they are exposed more to the clinical situations and complications and hence exhibit improved knowledge when compared to third year students . a significant difference was seen among the third year students when asked regarding the transmission of hbv through salivary contact . most of the third year students were not aware of the increased risk among dental professionals when compared to general population . since the students enter the clinical in the third year of study , proper immunization and education among them can minimize the early transmission of hbv among the dental students . the level of knowledge and compliance with infection control measures was poor among the students . attributable reasons could be inadequate training for infection control measures , inadequate supply of personal protective equipment , and carelessness . proper hand washing and use of barriers such as gloves , gowns , and mask are the main components of standard precautions which can minimize mucocutaneous exposures . reducing the manipulation of manual sharp instruments the use of puncture - resistant containers for sharp disposal is also an effective strategy . use of protective eye wares and face mask can help in preventing blood or saliva contact during the procedure . indirect transmission of hepatitis b virus can also occur through the dental instruments hence a proper method of sterilization needs to be educated among clinical students . vaccination against hepatitis b is recommended for all the dental students before they start their clinical phase and for susceptible dentists and dental auxiliary staff [ 14 , 15 ] . it is recommended that a policy be implemented for complete vaccination and health education for all dental students in first year in all dental colleges . however , antibody titers should be routinely checked among all vaccinated students because in some cases there are chances of nonresponse to the first series of vaccination . it is of prime importance for all dental schools to conduct talks and create awareness about hepatitis b infection . the study highlights the need for implementing separate course in dental curriculum on communicable diseases in the first year of dental school . at the end of our study the students were given a lecture and demonstration on various universal precaution methods which they should follow when treating any patients . firstly , all collected data were self - reported and therefore not verifiable . in particular , there was some inconsistency in the collected data ( only yes or no option was given for all questions ) , indicating that some of the reported information was unreliable . since it is a cross - sectional study the knowledge at that point of study lastly , because of the nonrandom sampling , our conclusions have limited generalizability to other dental college students in india . however the results of the study can be used as a baseline for enhancing the knowledge about infection control by conducting continuing dental education program . although dentists and dental students can be exposed to the human immunodeficiency virus ( hiv ) , hepatitis b virus ( hbv ) , and hepatitis c virus ( hcv ) in their work environment , this particular study evaluated the awareness only on hepatitis b virus since it is most commonly underdiagnosed and the risk of transmission is much higher compared to other communicable diseases . although the overall level of knowledge about viral hepatitis among the various years is fairly satisfactory , the students entering the third year of clinical study have less knowledge of hbv infection .

backgroundbiomass smoke is the leading cause of copd in developing countries such as turkey . in rural areas of turkey , females are more exposed to biomass smoke because of traditional lifestyles.aimthe aim of this study was to determine the adverse effects of biomass smoke on pulmonary functions and define the relationship between duration in years and an index ( cumulative exposure index ) with altered pulmonary function test results.participants and methodsa total of 115 females who lived in the village of kaizman ( a borough of kars located in the eastern part of turkey ) and were exposed to biomass smoke were included in the study . the control group was generated with 73 individuals living in the same area who were never exposed to biomass smoke.resultstwenty-seven ( 23.8% ) females in the study group and four ( 5.5% ) in the control group had small airway disease ( p=0.038 ) . twenty - two ( 19.1% ) females in the study group and ten ( 13.7% ) in the control group had obstruction ( p=0.223 ) . twenty ( 17.3% ) females in the study group who were exposed to biomass smoke had restriction compared with ten ( 13% ) in the control group ( p=0.189 ) . the duration needed for the existence of small airway disease was 16 years , for obstructive airway disease was 17 years , and for restrictive airway disease was 17 years . the intensity of biomass smoke was defined in terms of cumulative exposure index ; it was calculated by multiplying hours per day , weeks per month , and total years of smoke exposure and dividing the result by three.conclusionexposure to biomass smoke is a serious public health problem , especially in rural areas of developing countries , because of its negative effects on pulmonary functions . as the duration and the intensity of exposure increase , the probability of having altered pulmonary function test results is higher . hepatitis b virus transmission in a dental setting more commonly occurs due to inadequate / improper use of safety measures by the dentist . this particular study evaluated the hepatitis b virus infection related awareness among dental graduate students in a university dental college , india . a validated questionnaire regarding the awareness about hepatitis infection and various infection control measures was distributed among the students of different year of study in undergraduate bachelor dental graduate program . the data extracted were tabulated and analyzed . final year students showed an increased awareness when compared to third year students . there is need for improving the knowledge among the nonclinical students , mainly on transmission of virus through salivary contact . the overall awareness among the students is only fairly satisfying , which signifies the need for continued infection control education among the students .

a prospective molecular epidemiologic study of s. aureus was performed in a 12-bed icu at the university hospital in montpellier , france , during 5 months in 2011 . s. aureus nasal carriage was investigated at admission and weekly in 89 patients and monthly in 63 volunteer health care workers ( hcws ) . simultaneously , all s. aureus isolates from clinical samples were obtained from the hospital laboratory of bacteriology and clinical data were recorded . pneumonia was diagnosed on the basis of clinical , biologic , and radiologic criteria , and a colony count 10 cfu / ml in bronchoalveolar fluid culture or 10 cfu / ml in sputum cultures . bronchial colonization was defined as a colony count < 10 cfu / ml in sputum cultures in asymptomatic patients . random sampling of surfaces was performed monthly in all rooms of the icu ( 864 environmental sites ) . isolates were characterized by using multilocus sequence typing , double - locus sequence typing ( dlst ) , and accessory gene regulation ( agr ) typing . virulence genes and erma , ermc , ermt , and msra genes were screened for by using pcrs . during the survey period , the number of samples obtained ranged from 1 to 32 per patient and from 1 to 3 per hcw . of these samples , 125 s. aureus isolates ( 110 mssa and 15 mrsa ) were obtained from 33 patients , 26 hcws , and 36 environmental sites ; these isolates belonged to 28 sts and 12 ccs . among these 125 isolates , 12 isolates from 5 patients , 2 isolates from 2 hcws , and 15 isolates from 15 environmental sites belonged to cc398 ( figure 1 ; technical appendix ) . the 29 strains were mssa and belonged to st398 ( n = 25 ) or to a new st submitted to the mlst database ( http://www.mlst.net/ ) as st2658 ( n = 4 ) . st398 and cc398 were the most prevalent genotype and clonal complex identified : 25/125 ( 20% ) and 29/125 ( 23.2% ) isolates , respectively ( figure 2 ) . flowchart of selection for methicillin - sensitive staphylococcus aureus clonal complex ( cc ) 398 from intensive care unit , france , 2011 . principal clonal complexes ( ccs ) among 125 isolates of staphylococcus aureus from intensive care unit , france , 2011 . the prevalence of mssa cc398 carriage was 3.2% ( 2/63 ) in hcws and 5.6% ( 5/89 ) in patients . the prevalence of mssa cc398 infection was 2.25% ( 2/89 patients ) ( figure 1 ) . these patients were hospitalized during the same period ; nosocomial pneumonia developed after nasal colonization , and was associated with bacteremia in 1 case . demographic and clinical characteristics were similar in patients colonized or infected with mssa cc398 or with other genotypes ( table 1 ) . genotype cc398 was found more frequently in the icu environment ( 15/36 , 41.7% ) than in patients ( 5/33 , 15.2% ; 4.7 , p = 0.03 ) and hcws ( 2/26 , 7.7% ; 7.1 , p = 0.007 ) ( technical appendix ) . * copd , chronic obstructive pulmonary disease ; crf , chronic respiratory failure ; is , immunosuppressive therapy ; icu , intensive care unit ; saps ii , simplified acute physiology score ii . four strains belonged to the new st2658 , which differed from st398 by a synonymous mutation ( ag ) at position 198 of the pta gene . these 4 strains were isolated from nasal carriage samples ( n = 2 ) , bronchial colonization samples ( n = 1 ) , and pneumonia testing samples ( n = 1 ) from 2 patients hospitalized at the same time . all mssa cc398 strains were agr type 1 , spa type t571 ( determined by using dnagear software ; http://w3.ualg.pt/hshah/dnagear/ ) , and dlst type 144186 ( dlst spa 186 corresponding to spa type t571 ) . genes encoding panton - valentine leukocidin , toxic shock syndrome toxin 1 , and staphylococcal enterotoxin a were not detected . sensitivity testing of mssa cc398 isolates showed that all isolates were resistant to erythromycin and had an inducible macrolide lincosamide streptogramin b phenotype . in addition , resistance to penicillin and amoxicillin caused by -lactamase production was observed in 41.4% ( 12/29 ) of the strains . resistance to kanamycin , tobramycin , and gentamicin was observed in 24.1% ( 7/29 ) of the strains ; all 7 strains were isolated from environmental samples . analysis of genes encoding antimicrobial drug resistance identified the ermt gene in all the cc398 strains and a variable distribution of erma and ermc genes . * p , patient ; hcw , health care worker , e , environment . s , sputum ; n , nose ; bal , bronchoalveolar lavage fluid ; b , bloodstream . bl , -lactamase ( resistance to penicillin and amoxicillin ) ; imls , inducible macrolide lincosamide streptomycin b ; ktg , kanamycin , tobramycin , gentamicin . identification of mssa cc398 in hcws , patients without exposure to livestock , and the environment in an icu indicates emergence of this clone in a hospital in france . the prevalence of nasal carriage in hcws and patients was high ( 5.6% ) in the context of the icu , where these persons have frequent contact with each other . the small number of patients colonized or infected with s. aureus cc398 limits statistical comparison of the 2 groups and identification of risk factors for infection . despite the monocentric nature and the short period of the study , which limit extrapolation of our results to other settings , our study underlines the capacity of mssa cc398 to circulate among and between patients , hcws , and the icu environment . reported prolonged survival of bovine mssa st398 strain in the human nose after artificial inoculation , which suggested that competition with human strains might facilitate its spread ( 12 ) . identification of st2658 in 2 patients hospitalized at the same time reinforces the hypothesis of an increased capacity of transmission of this clonal complex between patients . person - to - person spread of mssa st398 has been reported within community households ( 8,10 ) and more recently in a hospital ( 11 ) and an urban jail ( 9 ) , in which a high proportion of detainees sharing a holding tank were colonized with mssa st398 ( 9 ) . these findings contrast with limited transmissibility of livestock - associated mrsa st398 , which is partially explained by molecular signatures of bacterial host adaptation identified only in the mssa st398 genome , such as different composition of adhesion genes that result in enhanced adhesion to human skin ( 10 ) . all strains were spa type t571 , which is the major spa type associated with mssa st398 ( 2,3,57 ) . there are other similarities between our strains and strains from china , spain , belgium , and the united states . ( 2,6,7,11 ) , such as agr type 1 , the presence of the ermt gene , tetracycline susceptibility , and macrolide lincosamide streptogramin b phenotype . in icus , the association of mssa cc398 with the icu environment suggests that this environment could play a role as a bacterial reservoir as described ( 14 ) . one hypothesis for such an association is the capacity to form a biofilm , which could be correlated with the s. aureus genetic background ( 15 ) . our findings emphasize potential hospital - adapted characteristics of s. aureus cc398 , which is supported by others studies ( 6,11 ) , and indicate that surveillance programs are needed to determine the role of this clonal complex , particularly in the hospital setting . distribution of staphylococcus aureus cc398 isolates from patients in intensive care unit , health care workers , and environmental sites , france , 2011 . a common reason for bringing an infant or child for medical visit is recurrent infections . this may refer to infections that are too great in number , too severe , or too long lasting ; that are associated with unusual complications ; or that fail to resolve with standard therapy . only 10% of such cases are due to deficiency in immunity either adaptive or innate . congenital neutropenia has an estimated frequency of two to three per million in the general population . elane ( elastase , neutrophil expressed ) gene related neutropenia includes congenital neutropenia and cyclic neutropenia , both of which are primary hematologic disorders characterized by recurrent fever , skin , and oropharyngeal inflammation ( ie , mouth ulcers , gingivitis , sinusitis , and pharyngitis ) , and cervical adenopathy . the diagnosis of elane - related neutropenia relies primarily on serial measurements of the absolute neutrophil count ( anc ) and clinical findings . molecular genetic testing of the elane gene , the only gene known to be associated with elane - related neutropenia , is available on a clinical basis . a 3-year - old girl was presented to the clinic with the complaint of recurrent fever in mid - september of the year 2009 . there was no significant birth history . during infancy , she had normal growth and no complaints suggestive of exocrine pancreatic insufficiency . on examination her stature was normal , there were no abnormality in skin pigmentation . on per abdominal examination investigations revealed anc of 639 cells / mm , that is , moderate neutropenia but other parameters were in normal range . she was advised biweekly follow - up for repeat cell count and monitoring for objective evidence of illness due to lack of authoritative past history , ie , history of recurrent fever prior to presentation in the clinic . she had to present everytime prior to the date of follow - up due to fever with either boils on skin or upper respiratory tract infection symptoms . it was then decided to put her on antibiotic prophylaxis and self - care advise including dental hygiene were explained to parents . follow - up visit throughout the period of 3 months showed consistent neutropenia with no cyclical variation in the count . she neither had any evidence of infection or disease that could result in neutropenia nor was on any drug that could result in the same . parents of the patient were explained about the possibility of various differential diagnosis that were probable . the report showed heterozygous mutation in exon2 at nucleotide position t201>a causing a codon change c55>s on polymerase chain reaction amplification followed by direct sequencing of all five exons and the promoter region of the neutrophil elastase ( ela2 ) gene . patient was started with filgrastim , recombinant methionyl human granulocyte - colony stimulating factor ( g - csf ) ( r - methug - csf ) on the weight - adjusted dosage schedule in february , 2010 . but in december , 2010 total count rose to 50 10 cells / l . histopathological examination of bone marrow specimen showed malignant transformation into acute myelogenous leukemia ( aml ) . most congenital neutropenia is diagnosed because of fever and severe infection in infants and young children . in 1956 , kostmann described congenital neutropenia ( agranulocytosis ) as an autosomal recessive disease occurring in an extended family in northern sweden . elane ( previously known as ela2 ) is the only gene known to be associated with elane - related neutropenia . its key physiologic role is in innate host defense , but it can also participate in tissue remodeling and possesses secretagogue actions important to local inflammatory responses . individuals have fever and recurrent skin and oropharyngeal inflammation , that is , mouth ulcers , gingivitis , sinusitis , pharyngitis , and cervical adenopathy . diarrhea , pneumonia , and deep abscesses in the liver , lung , and subcutaneous tissues are common . congenital neutropenia and cyclic neutropenia were initially thought to be distinct disorders ; however , following the discovery of the molecular basis of elane - related neutropenia , individuals with findings intermediate between these two phenotypes are also recognized . nonetheless , identification of the two phenotypes is helpful for diagnosis , prognosis , and management . diagnosis requires at least three ancs lower than 500 cells / mm obtained at least 3 months after the birth . bone marrow aspirate typically shows maturation arrest at the promyelocyte or myelocyte stage of neutrophil formation . the finding of an elane mutation in an individual with neutropenia establishes the diagnosis of elane - related neutropenia . common side effects of g - csf include bone pain and headache , splenomegaly , and osteoporosis . individuals with congenital neutropenia ( with or without an elane mutation ) who are treated with g - csf have approximately the same risk of the myelodysplasia syndrome / aml . the respective cumulative incidences 15 years after starting treatment with g - csf were 36% and 25% ( p=0.96 ) . the development of aml in our patient was within 10 months after starting g - csf . this is in contrast to the long 6-year observation period with 9% risk of transformation . hence , it is prudent to consider the aml as the predisposed malignancy associated with the natural history of these diseases . hematopoietic stem - cell transplant is the only alternative therapy for individuals with congenital neutropenia who are refractory to high - dose g - csf or who undergo malignant transformation . in our country , where infections are very common and the cost of g - csf treatment is high and self - financed , and also when a number of upcoming transplant centers are emerging , it is worthwhile to consider allogenic sibling stem - cell transplant , as one of the treatment modality , especially when there is an human leukocyte antigen ( hla)-matched sibling donor available . the cost - benefit ratio is much higher with the absence of long - term infectious complications and lesser infrequent hospital admissions . in our centre , we would start on prophylaxis with g - csf and then search for the hla - matched donor ( especially sibling ) for an allogenic transplant .

during testing for staphylococcus aureus in an intensive care unit in france in 2011 , we found that methicillin - sensitive s. aureus clonal complex 398 was the most frequent clone ( 29/125 , 23.2% ) . it was isolated from patients ( 5/89 , 5.6% ) , health care workers ( 2/63 , 3.2% ) , and environmental sites ( 15/864,1.7% ) . results indicate emergence of this clone in a hospital setting . we descibe the case of a girl of indian origin who presented with recurrent infections . the only abnormality detected in the armoury of the immune system was consistent neutropenia . mutation analysis revealed ela2 ( neutrophil elastase ) gene mutation that has been associated with severe congenital neutropenia phenotype . patient was treated with the granulocyte - colony stimulating factor ( g - csf ) as prevention of infectious manifestations along with appropriate measure to curb secondary complications . she showed poor response to the g - csf during stringent surveillance . after being on treatment for 1 year , she developed acute myelogenous leukemia as inherit complication of this disease .

gastric carcinoma is the fourth most common gastrointestinal cancer and has the second highest mortality rate among all gastrointestinal cancers . three critical measures improving patient prognosis are early diagnosis , radical surgery , and endoscopic surgery . although radical resection has been widely promoted , the post - surgery recovery is still unfavorable due to tnm stages . the prognosis of gastric cancer is also affected by patient biological factors and clinical pathological conditions , necessitating the identification of novel bio - markers for diagnosis and treatment . a widely accepted opinion is that oxidative stress ( os ) participates in pathogenesis of gastric cancer because it can damage dna and cause mucous injuries . nuclear factor e2-related factor 2 ( nrf2 ) , as a basic redox - sensitive bzip transcription factor , plays a role in intracellular anti - oxidation and phase ii detoxification through the up - regulation of various antioxidant response elements ( are ) genes via the keap1-cul3-dependent e3 ubiquitination pathway . under oxidative stress or chemical stimulus , nrf2 translocates into the nucleus , where it forms heterodimers with maf and binds onto the are sequence on the chromosome to activate the transcription of downstream genes , including antioxidants and phase ii detoxification enzymes [ 1214 ] . the importance of nrf2 resides in its protective roles against various human disease and pathological conditions , including cancer , neurodegenerative disease , cardiovascular dysfunction , inflammation , and pulmonary fibrosis , along with ameliorating aging [ 1519 ] , making nrf2 a beneficial transcriptional factor protecting the body from oxidative stress . in tumor tissues , therefore , nrf2 has a dual role in both preventing oncogenesis and protecting tumor cells from stress injury , making it a candidate factor for tumor growth and invasion . currently there is no available study regarding either expression or clinical significance of nrf2 in gastric carcinoma . this study therefore aimed to address this issue and further elucidate the clinical implication of anti - oxidative stress - related nrf2 expression . gastric cancer cells mkn74 , mnk45 , katoiii , and nugc4 ( cell institute of chinese academy of science , china ) were cultured in rpmi 1640 medium containing 10% fetal bovine serum ( fbs ) , 100 u / ml penicillin , and 100 g / ml streptomycin . cells collected by centrifugation were rinsed in phosphate - buffered saline ( pbs ) and extracted for total proteins by lysis buffer . cell nucleus / cytoplasmic fractions were separated using a test kit ( biovision , usa ) according to the manufacturer s instructions . denatured proteins were separated on 10% sds - page and transferred to pvdf membrane , which was blocked in 5% defatted milk powders overnight . mouse monoclonal anti - nrf2 antibody ( 1:500 , santa cruz , usa ) was used to incubate the membrane overnight . after rinsing in tris - buffered saline - tween 20 ( tbst ) , anti - mouse igg conjugated with horseradish peroxidase ( rd systems , us ) was used for a further 15-min incubation . the signal was finally developed using ecl - plus reagents and was visualized on x - ray films . the nucleus and cytoplasm fractions were labelled by anti - lamin b1 ( 1:1 000 , abcam , usa ) or anti- tubulin antibody ( 1:1000 , calbiochem , usa ) . all patients underwent surgical resection of stomach ( 57 cases of distal , 14 cases of proximal , 99 cases of total , and 5 cases of partial stomach resection ) with lymph node clearance between jan 2004 and dec 2013 in our hospital . there were 116 males and 59 females in the patient group , ages 3184 years ( average , 66 years ) . among all patients , 47 were at stage i when diagnosed , 33 were at stage ii , and 95 were at stage iii . histopathological typing showed 71 cases of differentiated ( papillary , high - differentiated , and moderate differentiated tubular adenocarcinoma ) tumors and 104 undifferentiated tumors ( including low differentiated adenocarcinoma , mucinous adenocarcinoma , and signet ring cell carcinoma ) . this study was pre - approved by the ethics committee of our hospital and we obtained written consent from all patients and their families . tumor tissues were first fixed in 10% formalin - pbs buffer , embedded in paraffin , sectioned ( 4-m thickness ) , and was mounted on glass slides . after de - waxing in xylene and re - hydration in gradient ethanol , endogenous peroxidase activity was quenched by 3% hydrogen peroxide in methanol . after gentle washing in pbs , antigen retrieval was performed using heated citrate buffers for 10 min . non - specific binding sites were further blocked in 10% fbs . anti - nrf2 antibody ( 1:200 , santa cruz , usa ) further detection was accomplished by streptavidin - biotin peroxidase ( sp ) kit ( nichirei , japan ) and was visualized by diaminobenzidine incubation . two independent blinded observers evaluated staining images . in brief , 10 representative regions within the tumor samples were selected . in each region , the expression was observed in 100 cells under a high - magnification microscope ( 400 ) . the undifferentiated region was preferred to differentiate tissues on the same slide . averaged nrf2 index was calculated based on the number of positive cells in each region . the expressional profile was described from both the positive rate and staining intensity , the latter classified as 0 , 1 + , and 2 + . we compared the nrf2 expression level in patients who underwent 5-fluorouracil ( 5-fu ) treatment . as only stage ii / iii patients were qualified for chemotherapy and patients with severe adverse effects were excluded , only 72 patients were recruited for the analysis of nrf2 expression and tumor chemoresistance . between - group comparisons were performed by chi - square , fisher s exact , or mann - whitney u test . the correlation between nrf2 expression and clinical indexes was analyzed by kaplan - meier curve , univariate / multivariate analysis , and log - rank test . gastric cancer cells mkn74 , mnk45 , katoiii , and nugc4 ( cell institute of chinese academy of science , china ) were cultured in rpmi 1640 medium containing 10% fetal bovine serum ( fbs ) , 100 u / ml penicillin , and 100 g / ml streptomycin . cells collected by centrifugation were rinsed in phosphate - buffered saline ( pbs ) and extracted for total proteins by lysis buffer . cell nucleus / cytoplasmic fractions were separated using a test kit ( biovision , usa ) according to the manufacturer s instructions . denatured proteins were separated on 10% sds - page and transferred to pvdf membrane , which was blocked in 5% defatted milk powders overnight . mouse monoclonal anti - nrf2 antibody ( 1:500 , santa cruz , usa ) was used to incubate the membrane overnight . after rinsing in tris - buffered saline - tween 20 ( tbst ) , anti - mouse igg conjugated with horseradish peroxidase ( rd systems , us ) was used for a further 15-min incubation . the signal was finally developed using ecl - plus reagents and was visualized on x - ray films . the nucleus and cytoplasm fractions were labelled by anti - lamin b1 ( 1:1 000 , abcam , usa ) or anti- tubulin antibody ( 1:1000 , calbiochem , usa ) . all patients underwent surgical resection of stomach ( 57 cases of distal , 14 cases of proximal , 99 cases of total , and 5 cases of partial stomach resection ) with lymph node clearance between jan 2004 and dec 2013 in our hospital . there were 116 males and 59 females in the patient group , ages 3184 years ( average , 66 years ) . among all patients , 47 were at stage i when diagnosed , 33 were at stage ii , and 95 were at stage iii . histopathological typing showed 71 cases of differentiated ( papillary , high - differentiated , and moderate differentiated tubular adenocarcinoma ) tumors and 104 undifferentiated tumors ( including low differentiated adenocarcinoma , mucinous adenocarcinoma , and signet ring cell carcinoma ) . this study was pre - approved by the ethics committee of our hospital and we obtained written consent from all patients and their families . tumor tissues were first fixed in 10% formalin - pbs buffer , embedded in paraffin , sectioned ( 4-m thickness ) , and was mounted on glass slides . after de - waxing in xylene and re - hydration in gradient ethanol , after gentle washing in pbs , antigen retrieval was performed using heated citrate buffers for 10 min . non - specific binding sites were further blocked in 10% fbs . anti - nrf2 antibody ( 1:200 , santa cruz , usa ) further detection was accomplished by streptavidin - biotin peroxidase ( sp ) kit ( nichirei , japan ) and was visualized by diaminobenzidine incubation . two independent blinded observers evaluated staining images . in brief , 10 representative regions within the tumor samples were selected . in each region , the expression was observed in 100 cells under a high - magnification microscope ( 400 ) . the undifferentiated region was preferred to differentiate tissues on the same slide . averaged nrf2 index was calculated based on the number of positive cells in each region . the expressional profile was described from both the positive rate and staining intensity , the latter classified as 0 , 1 + , and 2 + . we compared the nrf2 expression level in patients who underwent 5-fluorouracil ( 5-fu ) treatment . as only stage ii / iii patients were qualified for chemotherapy and patients with severe adverse effects were excluded , only 72 patients were recruited for the analysis of nrf2 expression and tumor chemoresistance . between - group comparisons were performed by chi - square , fisher s exact , or mann - whitney u test . the correlation between nrf2 expression and clinical indexes was analyzed by kaplan - meier curve , univariate / multivariate analysis , and log - rank test . as shown by western blotting in both nucleus and cytosolic fractions , nrf2 protein is mainly expressed in the nucleus , with minimal expression in the cytoplasm ( figure 1 ) . ihc staining obtained results consistent with those in the abovementioned western blotting , as the nrf2 signal localizes in the nucleus ( figure 2 ) . we calculated the staining scored as described in the methods section , and classified those with scores higher than 100 into the nrf2-positive group . in all 175 patients , 108 were classified into the positive group ( 61.7% ) and the other 67 were classified into the negative group ( 38.3% ) . further correlation analysis between nrf2 expression and clinical indexes significant correlations were discovered between nrf2-positive expression and patient sex , tumor volume , infiltrative depth , lymph node metastasis , lymph tube invasion , and tnm staging . among all 72 patients who underwent 5-fu therapy , there were 59 who were nrf2-positive and 13 who were nrf2-negative . among those 59 nrf2-positive patients , a total of 43 patients ( 72.9% ) developed 5-fu resistance , which occurred in only 5 of the 13 nrf2-negative patients ( 38.5% ) . using kaplan - meier method , we found that the overall survival rate in nrf2-positive patients was significantly lower than that in nrf2-negative patients ( 61% vs. 79% , p<0.01 , figure 3 ) . significant correlation factors of patient survival rate include invasive depth , tumor size , lymph node metastasis , tnm stage , and lymph tube infiltration , by univariate analysis . a further multivariate analysis indicated lymph node metastasis as an independent prognostic index ( table 3 ) . as shown by western blotting in both nucleus and cytosolic fractions , nrf2 protein is mainly expressed in the nucleus , with minimal expression in the cytoplasm ( figure 1 ) . ihc staining obtained results consistent with those in the abovementioned western blotting , as the nrf2 signal localizes in the nucleus ( figure 2 ) . we calculated the staining scored as described in the methods section , and classified those with scores higher than 100 into the nrf2-positive group . in all 175 patients , 108 were classified into the positive group ( 61.7% ) and the other 67 were classified into the negative group ( 38.3% ) . further correlation analysis between nrf2 expression and clinical indexes significant correlations were discovered between nrf2-positive expression and patient sex , tumor volume , infiltrative depth , lymph node metastasis , lymph tube invasion , and tnm staging . among all 72 patients who underwent 5-fu therapy , there were 59 who were nrf2-positive and 13 who were nrf2-negative . among those 59 nrf2-positive patients , a total of 43 patients ( 72.9% ) developed 5-fu resistance , which occurred in only 5 of the 13 nrf2-negative patients ( 38.5% ) . using kaplan - meier method , we found that the overall survival rate in nrf2-positive patients was significantly lower than that in nrf2-negative patients ( 61% vs. 79% , p<0.01 , figure 3 ) . significant correlation factors of patient survival rate include invasive depth , tumor size , lymph node metastasis , tnm stage , and lymph tube infiltration , by univariate analysis . a further multivariate analysis indicated lymph node metastasis as an independent prognostic index ( table 3 ) . as an antioxidant , nrf2 can protect normal cells from oxidative stress injuries , thereby forming a self - protective mechanism . the over - expression of nrf2 and its downstream genes in various tumors , however , raised the potency of nrf2 in facilitating survival and proliferation of cancer cells [ 2026 ] . our study for the first time reports that a close relationship exists between nrf2 expression in gastric carcinoma cell nucleus and patient clinical features . the prominent expression of nrf2 in nucleus of gastric cancer cells from both in vivo and in vitro samples suggests a persistent expression of nrf2 may cause production of antioxidants , which further endow cancer cells with elevated anti - reactive oxygen species ( ros ) activity . this proposed mechanism has been reported by ma et al . , who found higher nuclear nrf2 levels and downstream anti - oxidative proteins in more advanced cervical cancer tissues . therefore , it is likely that gastric cancer cell nuclear nrf2 level is related with tumor malignancy . ihc staining results showed elevated nrf2 expression in gastric cancer tissues . among all patients , 61.7% were nrf2-positive , higher than those in non - small cell lung cancer ( nsclc ) ( 26% ) or gall bladder cancer ( 23% ) . the survival rate - related factors include tumor invasive depth , tumor size , lymph node metastasis , tnm stage , and lymph tube infiltration , in agreement with a gall bladder cancer study . recent study in gastric carcinoma found consistent effects of nrf2 expression on the prognosis , but only with immune reactivity in cytoplasm rather than the nucleus , probably due to the use of different antibodies . various studies have confirmed the importance of cytoplasmic - nuclear translocation of nrf2 for exerting its anti - oxidative activity . persistent over - expression of nrf2 may protect cancer cells from ros injuries because it can work as an anti - oxidant . therefore , it is necessary to quantify nuclear but not cytoplasmic nrf2 proteins in gastric cancer tissues . although nrf2-positive is not included in multivariate analysis as an independent prognostic evaluating factor , our results showed that lymph node metastasis can significantly affect nrf2-positive rates as a marker for unfavorable prognosis . previous studies in nsclc and gall bladder cancer all supported existence of relationships between nrf2 positive expression and unfavorable prognosis , suggesting the utility of nrf2 as a prognostic index for evaluating the postoperative survival rate of patients . various studies have shown that nrf2 can decrease the survival rate via its facilitation on tumor cell resistance against radio-/chemo - therapy [ 2934 ] . this study found a higher rate of 5-fu resistance in nrf2-positive tumors ; therefore , the evaluation of nrf2 in gastric cancer patients may help to optimize the chemotherapy plan . the genetic silencing or functional inhibition can suppress activity of nrf2-modulated oxidase , including glutathione , thioredoxin , and mercaptan sulfur , leading to the recovery of tumor cell sensitivity to chemo-/radio - therapy . there have been clinical trials supporting this mechanism , such as the sensitization of cancer cells against alkylation drugs by nrf2 inhibition or the inhibition of tumor growth by the combination of nrf2 gene knockdown and cisplatin application . therefore , the next generation of chemotherapy plans involving modulating nrf2-related anti - oxidative functions may provide more satisfactory treatment efficacy and improve patient survival and prognosis . this study demonstrated a positive relationship between nrf2 expression and invasiveness of gastric carcinoma , suggesting the utility of nrf2 as a prognostic index . copy number variation ( cnv ) is a structural genomic variation of the human genome that may either be inherited or caused by de novo mutation . cnvs can range in size from kilobases ( kbs ) to several megabases ( mbs ) that have not been identified by conventional chromosomal analysis . however , recent technology of genome - wide analysis such as comparative genomic hybridization ( cgh ) has led to the discovery of extensive genomic structural variation [ 13 ] . a recent report using microarray technology revealed that as much as 12% of the human genome are variable in copy number . these known cnvs are available from the interactive web - based database decipher ( database of chromosomal imbalance and phenotype in humans using ensembl resources , http://decipher.sanger.ac.uk/ ) . the decipher database is a consortium comprised of an international network of more than 100 centers and has uploaded more than 2000 cases ( current statistics can be found on the decipher homepage ) . de novo mutations are more likely to contribute to the development of sporadic genomic disorders [ 6 , 7 ] . in psychiatric disorders , asd and schizophrenia , extension of genome - wide association studies ( gwas ) have led to the discovery of both inherited and de novo sporadic cnvs . such cnvs resulted in altering gene dosage and dosage - sensitive gene expression , which may contribute to these disorders complexities . these human genetics studies have detected several cnvs ( e.g. , 1q21 , 3q29 , 10q26 , 11p14 , 15q11 , 15q13 , 16p13 , 17p12 , and 22q11 ) . this discovery suggests an important role for the strict regulation of gene dosage in asd and schizophrenia . to understand psychiatric disorders , while it is difficult to model human psychiatric phenotypes in animals ( e.g. , hallucinations and delusions characteristic of schizophrenia that are human specific ) , animal models may contribute to the elucidation of brain anatomy , behavioral characteristics , and molecular mechanisms that reflect aspects of human phenotypes . although there is a strong association between genetic rearrangement and psychiatric disorders ( e.g. , asd and schizophrenia ) , valid animal models that reflect etiology are rare . several efforts have been made to generate mouse models of psychiatric disorders by conventional gene targeting , conditional gene targeting , and point mutation by chemical mutagens . but these techniques are not enough to reflect complex human genomic rearrangements , such as large deletions , inversions , and duplications . in this regard , the cre / loxp - based chromosome engineering technique is useful to generate this kind of complex genomic rearrangements in the mouse genome . by using this chromosome engineering technique , we can accomplish cnv - based unbiased animal models of psychiatric disorders . in this paper , we focus on animal model of asd ( and schizophrenia ) which was generated by chromosome engineering , principle of this technology , and discuss for future directions . genetic abnormalities such as point mutations , deletions , duplications , inversions , and translocations can be induced by exposure to x - ray radiation , chemical mutagens ( e.g. , n - ethyl - n - nitrosourea ( enu ) ) , conventional gene - targeting , or chromosome engineering . the chemical mutagen , enu , induces single - base - pair substitutions in the genome causing mutations with partial functions [ 17 , 18 ] . animal models containing genes with point mutations can be used to reveal the gene 's functional domain in vivo . conventional gene targeting ( replacement ) is used to disrupt a gene ( inserting markers or reporters ) to determine a gene 's function . conditional gene - targeting utilizing the cre / loxp and flp / frt system allows spatial and temporal control of gene expression . chromosome engineering is based on cre / loxp technology , which can induce chromosome rearrangements ( deletions , duplications [ 10 , 20 , 21 ] , and inversions [ 22 , 23 ] ) in the mouse genome ( figure 1 ) . targeting vectors can be targeted in two orientations that result in deletion , duplication , or inversion . each targeting vector has a loxp site and drug selection marker , neomycin resistance ( neo ) , or puromycin - resistant gene ( puro ) . two loxp sites are sequentially inserted by each targeting vector into the mouse embryonic stem ( es ) cell genome . the vectors are manipulated by hypoxanthine phosphoribosyl transferase ( hprt ) expression following cre recombinase expression in es cells . clones which carry the desired chromosomal rearrangement are identified by various methods : drug selection by hypoxanthine - aminopterin - thymidine ( hat ) media , genomic southern blot analysis , fluorescent in situ hybridization ( fish ) , and cgh ( comparative genomic hybridization ) array . although cgh array can not identify structural chromosome aberrations such as balanced reciprocal translocations and inversions , this technique is a powerful tool to detect cnvs from genome . to inactivate a target gene or locus by chromosome engineering the mutagenic insertion and chromosome engineering resource ( micer ) ( http://www.sanger.ac.uk/resources/mouse/micer/ ) was developed by dr . allan bradley 's group , the wellcome trust sanger institute and is useful as a gene - targeting vectors resource . these ready to use targeting vectors can be accessed through the ensembl mouse genome browser ( http://www.ensembl.org/index.html ) . it is important to note that these targeting vectors use an insertion vector system rather than a replacement vector system . given the same length of homologous sequence insertion vectors have a ninefold higher targeting efficiency than replacement vectors . in spite of a strong association between asd ( and schizophrenia ) and cnv , animal models of cnv that reflect human genomic rearrangement are few . these animal models were generated by chromosome engineering and have several psychotic phenotypes similar to those seen in patients with genomic rearrangement ( table 1 ) . in this section we focus on 15q11 - 13 , 16p11.2 , and 22q11 locus , which are well - known copy number variant linked to asd ( or / and schizophrenia ) . human chromosome region , 15q11 - 13 , is a complicated region that contains -aminobutyric acidreceptor a ( gabaa receptor ) clusters and several imprinting genes . in addition to these genes , this locus includes noncoding small nucleolar rnas ( snornas ) that are located between snurf - snrpn and ube3a , which are paternally expressed and brain specific [ 27 , 28 ] . prader - willi syndrome ( pws ) and angelman syndrome ( as ) are affected by changes in the 15q11 - 13 locus . major clinical features of pws include low birth weight , short stature , small hands and feet , severe hypotonia , feeding difficulties , obesity associated with hyperphagia starting in early childhood , mild to moderate mental retardation , and learning and behavioral problems including obsessive - compulsive disorder and autism [ 29 , 30 ] . as patients exhibit developmental delay , gait ataxia , balance disorder , frequent laughter / smiling , easily excitable personality , hyperactivity , speech impairment , microcephaly , seizures , epilepsy , and abnormal eeg ( electroencephalogram ) . additionally , as patients often exhibit socialization and communication deficits , which are diagnostic criteria for asd [ 32 , 33 ] . duplication of the 15q11 - 13 locus was first reported as a partial trisomy of chromosome 15 , and then two individuals with autistic disorder were reported . this locus has been known as the most frequent cytogenetic abnormality in asd [ 36 , 37 ] . generally patients with 15q11 - 13 duplication show hypotonia , delay in motor skills and language development , epilepsy , and cognitive and learning problems . recently , michelson et al . reported a patient with severe intractable epilepsy who has familial partial trisomy 15q11 - 13 inherited from a mother who has schizophrenia . autistic phenotype associated with 15q11 - 13 duplication , usually believed that maternal origin , ube3a is involved [ 3946 ] . although maternal locus supposed to critical , paternally inherited patients had also developmental delay [ 44 , 4649 ] . clinical reports have been accumulating but no mechanism has been addressed . to address this question , nakatani et al . this mouse was generated by chromosomal engineering based on the cre / loxp system , and it has a 6.3 mb duplicated locus in mouse chromosome 7c which is highly similar to human 15q11 - 13 ( figure 2(a ) ) . gene expression analysis revealed that paternally expressed genes , both ndn and snrpn , were twofold higher in paternally inherited mice ( patdp/+ ) than wild - type ( wt ) mice . similarly , maternally expressed gene ube3a was twofold higher in maternally inherited mice ( matdp/+ ) than wt mice . monoamine levels in patdp/+ adult mice , serotonin ( 5-ht ) , and their metabolites 5-hydroxyindoleacetic acid ( 5-hiaa ) were significantly downregulated in the midbrain and olfactory bulb . also 5-ht content in developmental stage from postnatal 1 to 3 weeks in patdp/+ brain regions ( cortex , hippocampus , cerebellum , midbrain , hypothalamus , pons , and medulla ) was downregulated . this indicates 5-ht signaling during the developmental stage was significantly impaired in the brains of patdp/+ mice . 5-ht influences not only mental condition ( mood , social behavior , appetite , aggression and sleep ) but also normal development of the central nervous system [ 5052 ] . in addition to this , abnormal 5-ht levels have been found in asd patient blood cells . treatment with serotonin reuptake inhibitors ( ssris ) have shown moderate success in recovering behaviors . behavioral tests revealed that patdp/+ mice display autistic behaviors such as less social interaction in the three - chamber social interaction test , abnormal ultrasonic vocalizations ( usvs ) in postnatal developing pups separated from their dams , and behavioral inflexibility in the morris water maze and barnes maze . the phenotypes seen in patdp/+ mice indicate that these mice have impaired behaviors that include social interaction , communication , restricted interest , and resistance to change . furthermore , patdp/+ mice showed anxiety - related phenotypes : decreased locomotor and exploratory activities in the open field and y - maze test , and long latencies in novelty suppressed feeding test . these anxiety - related phenotypes frequently accompany autistic symptoms in humans [ 58 , 59 ] . also the marble burying test , which is a useful test for the study of anxiety , obsessive - compulsive disorder ( ocd ) , and neophobia , found that the number of buried marbles was significantly low in patdp/+ mice . deletion or duplication of the chromosome 16p11.2 locus was observed in nearly 1% of multiplex families with asd . meta - analysis of patients with asd and/or developmental delay estimated that 16p11.2 locus deletion is associated with a 38.7-fold increase in the odds of asd / developmental delay . on the other hand , 16p11.2 locus duplication is associated with a 20.7-fold increase in the odds of asd / developmental delay [ 6063 ] . in addition to these , 16p11.2 deletion is associated with obesity , and duplication is associated with schizophrenia as well as asd [ 60 , 66 ] . notably , a brain anatomical abnormality ( abnormal head size ) has been reported to be associated with this locus . for instance , patients with the 16p11.2 deletion had statistically significant macrocephaly and those with duplication had microcephaly . a mouse model of human 16p11.2 deletion ( df/+ ) as well as duplication ( dp/+ ) has been reported ( figure 2(b ) ) . this locus includes 27 genes , spn , qprt , c16orf54 , kif22 , maz , prrt2 , c16orf53 , mvp , cdipt , sez6l2 , asphd1 , kctd13 , loc124446 , hirip3 , ccdc95 , doc2a , fam57b , aldoa , ppp4c , ypel3 , gdpd3 , mapk3 , and coro1a . young df/+ mice ( before weaning ) tend to be smaller than wt siblings , but as adults they are almost the same size as wt siblings and look healthy . interestingly , 16p11.2 cnv mice , df/+ and dp/+ mice have opposite phenotypes . in a novel environmental cage , df/+ mice displayed longer distance traveled and time spent walking as compared with wt mice . in contrast , dp/+ mice traveled a shorter distance and spent less time walking as compared with wt mice . additionally , df/+ mice were significantly active in both dark and light period . these results indicate that 16p11.2 locus affects not only physical activity but also diurnal activity and sleeping related symptoms . also , a brain anatomical study using magnetic resonance imaging ( mri ) identified several regional changes in 16p11.2 cnv mice . for instance , df/+ mice showed increased volume of several brain regions ( percentage of total brain volume ) : forebrain , superior colliculus , fornix , hypothalamus , mammillothalamic tract , medial septum , midbrain , and periaqueductal grey . these brain regional volumetric changes were more significant between df/+ and dp/+ than betweendf/+ and wt mice . microdeletion of chromosome 22q11 is found in 1 out of every 4000 live births , making it one of the most common interstitial deletions . this 22q11.2 microdeletion causes craniofacial , cardiovascular abnormalities , immunodeficiency , hypocalcaemia , short stature , and cognitive dysfunctions [ 6971 ] . microdeletion of this region accounts for 1 - 2% of the cases of people with schizophrenia [ 72 , 73 ] . also , this locus accounts for up to 1 - 2% of cases of sporadic schizophrenia [ 7476 ] . volumetric reduction in total brain volume includes cortical regions ( e.g. , frontal , parietal , temporal , and occipital lobes ) , hippocampus , and cerebellum [ 7786 ] . however , inconsistency in these neuroimaging reports may be due to the small numbers of subjects used and differences in methodology . yet these neuroanatomical reports are informative because some abnormalities are consistent with phenotypes of those who have non-22q11.2 ds - associated schizophrenia . the majority of deletions in this locus are 3 mb deletions ( 90% of the cases ) , but 1.5 mb deletions ( < 10% of the cases ) contain 28 known genes which include critical genes and increased risk of mental disorders [ 73 , 87 ] . animal models of the human 22q11.2 deletion were generated by 2 groups , and both groups used chromosome engineering [ 14 , 88 ] ( figure 2(c ) ) . these mouse models , df(16)a and lgdel/+ , include 1.5 mb critical regions , and both of them display several behavioral abnormalities , such as deficits in working memory , sensorimotor gating , and fear conditioning [ 14 , 8992 ] . working memory deficits are becoming one of the main features of patients with schizophrenia , thus these animal models are supposed to reflect some aspects of 22q11.2 ds syndrome phenotype . in addition to behavioral abnormalities in this mouse , diminished 22q11 locus dosage disrupts cortical neurogenesis , interneuron migration , dendritic complexity , and formation of excitatory synapses . although , several interesting phenotypes have been reported in this mutant mouse , there are no studies published about brain structural abnormalities even though several brain abnormalities have reported in human studies . these brain anatomical changes and molecular mechanisms that underlie these phenotypes will be interesting to elucidate and will be addressed by using brain imaging techniques . application of new technologies , such as comparative genomic hybridization ( cgh ) and next - generation sequencing , will reveal more additional genomic rearrangements related to psychiatric disorders . thus , to analyze both phenotypes and underlying molecular mechanisms that originate from genetic rearrangements , animal models will be a powerful tool . in this context reported that they generated several hundred mice and embryos which have one loxp and lacz site at a random genomic positions that inserted by sleeping beauty - based transposition system . these lines are mapped in transposon and recombinase associated chromosomal engineering resource database ( tracer , http://tracerdatabase.embl.de/fmi/iwp/res/iwp_home.html ) . it is an orthodox approach to narrow down the region by systematically insertion of loxp combining the existed lines such as above tracer . generating bacterial artificial chromosome ( bac ) transgenic mice is another way to identify critical genes . bac transgenes inserted to the genome faithfully recapitulate chromosomal endogenous gene expression , since bac transgenic mice may appropriate animal model of gene duplication . also transient overexpressing ( or knockdown ) each transcript in developmental brain is possible strategy . identified a responsible gene kctd13 in 16p11.2 locus which causes brain malformation by using zebrafish . use of these technologies in generating valid and etiology - based animal model of psychiatric disorders will contribute to the development of drugs against disorders and elucidation of molecular mechanisms that underlie these psychiatric disorders .

backgroundnuclear factor e2-related factor 2 ( nrf2 ) plays an anti - oxidative and phase ii detoxification function via its up - regulation on various antioxidant response elements ( are ) genes . nrf2 can protect both normal and cancer cells from damages of cell stress , thereby exerting a critical role in the development of cancer . the expression and significance of nrf2 in gastric cancer , however , has not been reported . this study thus aimed to investigate the expression of nrf2 in gastric cancer tissues via immunohistochemical ( ihc ) staining.material/methodsgastric carcinoma tissues from a total of 175 patients during surgical resection were examined for nfr2 expression profiles using ihc staining on paraffin - embedded slides . between - group - comparisons were performed by chi - square , fisher s exact , or mann - whitney u test . the correlation between nfr2 expression and clinical indexes was further analyzed by kaplan - meier test , univariate / multivariate analysis , and log - rank test.resultsnrf2 is mainly expressed in nuclei of gastric carcinoma tissues , with significant correlation with clinical indexes , including tumor size , invasive depth , lymph node metastasis , and invasion . patients with nrf2-positive expression had significantly lower survival rates compared to those in the negative group ( p<0.01 ) , with chemo - resistance against 5-fluorouracil ( 5-fu ) ( p<0.05).conclusionsnrf2 expression is positively correlated with invasive gastric cancer , suggesting its utility as a predictive index for unfavorable prognosis . the development of genetic technologies has led to the identification of several copy number variations ( cnvs ) in the human genome . genome rearrangements affect dosage - sensitive gene expression in normal brain development . there is strong evidence associating human psychiatric disorders , especially autism spectrum disorders ( asds ) and schizophrenia to genetic risk factors and accumulated cnv risk loci . deletions in 1q21 , 3q29 , 15q13 , 17p12 , and 22q11 , as well as duplications in 16p11 , 16p13 , and 15q11 - 13 have been reported as recurrent cnvs in asd and/or schizophrenia . chromosome engineering can be a useful technology to reflect human diseases in animal models , especially cnv - based psychiatric disorders . this system , based on the cre / loxp strategy , uses large chromosome rearrangement such as deletion , duplication , inversion , and translocation . although it is hard to reflect human pathophysiology in animal models , some aspects of molecular pathways , brain anatomy , cognitive , and behavioral phenotypes can be addressed . some groups have created animal models of psychiatric disorders , asd , and schizophrenia , which are based on human cnv . these mouse models display some brain anatomical and behavioral abnormalities , providing insight into human neuropsychiatric disorders that will contribute to novel drug screening for these devastating disorders .

osteoporosis is a systemic skeletal disease characterized by reduced bone mineral density and microarchitectural deterioration , compromising bone strength and increasing risk of fractures . magnetic resonance imaging ( mri ) is an ionizing radiation - free imaging modality , the application of which could be beneficial in the elderly population to monitor incidence , progression , and therapy of osteoporosis . due to the lack of signal from the bone matrix in conventional mri sequences , high - resolution mri of the trabecular bone has been previously accomplished relying on the bone marrow signal surrounding the bone matrix [ 2 , 3 ] . the vertebral bodies , one of the most important fracture sites , are filled with red marrow , which is a mixture of haematopoietic red blood cells and fat cells . despite recent progress [ 4 , 5 ] , direct high - resolution imaging of the trabecular bone has been limited on distal sites ( e.g. , radius , tibia , or calcaneus ) and its application in red marrow regions remains technically challenging . previous mri investigations have studied alternative indirect measures of trabecular bone quantity and quality in red marrow regions , including approaches aiming to measure primarily bone marrow t2 [ 6 , 7 ] . another property of bone marrow , which has recently gained significant attention due to its potential association with bone loss pathophysiology , is its fat content [ 810 ] . mr investigations employing single - voxel magnetic resonance spectroscopy ( mrs ) have recently shown the increase of vertebral bone marrow fat content with age in large scale in vivo studies [ 12 , 13 ] . in addition to age - related change of bone marrow fat content , recent in vivo studies have shown that an increase in bone marrow fat content is associated with a decrease in bone mineral density ( bmd ) [ 7 , 1420 ] . furthermore , there have been a limited number of studies analyzing transiliac bone biopsy samples showing an association between bone marrow adiposity and bone microstructure . additionally , it has been shown that imaging - based trabecular bone microstructure and texture parameters ( using primarily computed tomography ( ct ) and its variants ) can add significant information beyond bmd on predicting bone strength [ 23 , 24 ] . based on the negative association between bmd and vertebral bone marrow fat content , previous studies have recently proposed mrs - based bone marrow fat quantification as a potential noninvasive biomarker for prediction of fracture risk . however , many of these studies have used dual x - ray absorptiometry ( dxa ) for measurement of bmd , including therefore contributions from both the trabecular and the cortical bone compartments in the reported bmd measurements [ 7 , 1517 ] . in addition , there has been no previous groundwork on directly investigating the relationship between bone marrow fat content and biomechanical strength . determining the relationship between bone marrow fat content and trabecular bone microstructure parameters would require sampling larger specimens than those obtained with biopsy . a multimodality ex vivo study in bone specimens combining ct and mrs measurements with biomechanical testing would be required to study the aforementioned relationships . therefore , the purpose of the present pilot study was to investigate the correlation of mrs - based proton density fat fraction with bmd / trabecular bone microstructure parameters obtained from multidetector ct ( mdct ) measurements and bone strength determined from biomechanical testing , using human vertebral cadaveric specimens . ten vertebrae between thoracic vertebrae 5 and 10 ( t5t10 ) were harvested from four fresh human cadavers ( 1 woman and 3 men ; mean age standard deviation ( sd ) of 58 12 years ) . donors had no history of pathological bone changes other than osteoporosis ( i.e. , bone metastases , hematological , or metabolic bone disorders ) . the donors had dedicated their bodies for educational and research purposes to the local institute of anatomy prior to death , in compliance with local institutional and legislative requirements . written informed consent the surrounding muscle , fat tissue , and intervertebral discs were completely removed from the vertebrae . each vertebra was embedded in resin ( rencast isocyanat and polyol , huntsman group , bad sckingen , germany ) up to 2 mm above and below their vertebral endplates for the purpose of biomechanical testing . the resin fixation was performed with parallel alignment of the upper and lower endplate of the vertebrae with the outer surface of the resin chock to guarantee strict axial loading conditions of the vertebrae during the uniaxial biomechanical test . for the purpose of conservation , all vertebrae were stored in the freezer at 4 celsius during the study and degassed in sodium chloride solution at least 3 h before imaging to prevent air artifacts . the vertebrae were sealed in vacuum plastic boxes filled with sodium chloride solution during imaging . multidetector computed tomography ( mdct ) images of the vertebrae were acquired by using a whole - body 256-row ct scanner ( ict , philips medical care , best , netherlands ) . scan parameters were a tube voltage of 120 kvp , a tube load of 585 mas , an image matrix of 1024 1024 pixels , and a field of view of 150 mm . the real spatial resolution was 230 230 600 m as determined at 50 of the modulation - transfer - function . a dedicated calibration phantom ( mindways osteoporosis phantom , san francisco , ca , usa ) was placed in the scanner mat beneath the vertebrae . mdct images were transferred to a remote linux workstation and loaded into an in - house developed program based on idl ( interactive data language , research systems , boulder , co , usa ) . then , transverse cross - sectional area was determined in each section to obtain the mean and minimum transverse cross - sectional area of each vertebra . second , the twenty most central slices displaying the vertebra equidistant to its endplates were identified ( figure 1(a ) ) . similar to the mrs box ( as outlined below ) , rectangular regions of interest ( rois ) with an area of 12 12 mm were manually placed in the center of the vertebra in the selected twenty slices of the mdct images . lastly , rois were drawn in the phases of the calibration phantom in the mdct images . mean bmd in the rois was calculated by converting the pixel attenuations in hounsfield units [ hu ] into bmd values in calcium hydroxyapatite [ mg / cm ] by using the calibration phantom . similar to previous studies , 200 mg / cm calcium hydroxyapatite was identified as optimized global threshold [ 25 , 26 ] . two morphometric parameters were calculated in the rois in analogy to standard histomorphometry using the mean intercept length method : bone volume divided by total volume ( bv / tv ) and trabecular number ( tbn ; [ mm ] ) . in addition , fractal dimension ( fd ) as texture measurement of the trabecular bone microstructure was determined in the mdct images using a box counting algorithm as previously described . the vertebrae were scanned on a 3 t whole - body scanner ( ingenia , philips healthcare , best , netherlands ) using an 8-channel extremity coil . the mr exam consisted of two fat suppressed proton density- ( pd- ) weighted turbo spin - echo ( tse ) sequences ( one mimicking the sagittal anatomical orientation and one the axial anatomical orientation of the vertebrae ) and one single - voxel mrs sequence . sequence parameters for the tse sequence were te / tr = 40/3165 ms , tse factor = 11 , fov = 130 130 , 25 slices , slice thickness = 1.5 mm , and bw = 156.7 hz / pixel . based on the specimen geometry outlined in the pd - weighted sequences ( figure 1(b ) ) , a voxel was selected in the center of the vertebral body to perform single - voxel ( 12 12 12 mm ) mrs using a stimulated echo acquisition mode ( steam ) sequence with parameters : tr = 6 s ( long tr to remove any t1 effects ) , te = 12/15/20/25 ms ( steam with short tes to reduce j - coupling effects ) , 10 averages per te , 2 phase cycles , 4096 data points , 5 khz acquisition bandwidth , no water suppression , and no regional saturation bands . the voxel was positioned so that chemical shift displacement effects due to the finite bandwidth of the employed rf pulses used in the mrs voxel localization were minimized . spectra were fitted using gaussian line shapes and frequency - based methods based on in - house matlab ( mathworks , natick , ma ) routines . figure 1(d ) shows a typical bone marrow fat spectrum with fat peaks observed at spectral locations at 0.9 , 1.30 , 1.59 , 2.00 , 2.25 , 2.77 , 4.2 , 5.19 , and 5.31 ppm . the letters a , d , and e were assigned to peaks at 0.9 ppm ( (ch2)n ch3 ) , 2.77 ppm ( ch = ch ch2ch = ch ) , and 4.2 ppm ( ch2o co ) , respectively . the letter b was assigned to the superposition of peaks at 1.30 ppm ( (ch2)n ) and 1.59 ppm ( co ch2ch2 ) , the letter c was assigned to the superposition of peaks at 2.00 ppm ( ch2ch = ch ch2 ) and 2.25 ppm ( co ch2ch2 ) , and the letter f was assigned to the superposition of peaks at 5.19 ppm ( ch o peak fitting was performed by constraining the area of peaks e and f at a given ratio of peak a + b , based on the bone marrow triglyceride chemical structure determined previously . a common linewidth was assumed for all fat peaks and independent linewidth values were fitted for the two water peaks , resulting in a total number of three linewidths as free variables . fat peak locations were allowed to vary by 0.05 ppm and water peak locations were allowed to vary by 0.50 ppm . t2 correction was then performed using nonlinear least squares fitting , assuming the same t2 relaxation time value for all fat peaks and a different value for the water t2 relaxation time . the derived proton density fat fraction was determined as the ratio of all the fat peaks ( a , b , c , d , e , and f ) area with the sum of all the fat peaks and the narrow ( long t2 ) water peak area ( i.e. , excluding the broad - short t2 water peak area ) . the resin embedded vertebrae were fixed in a mechanical testing system ( wolpert werkstoffprfmaschinen ag , schaffhausen , switzerland ) . firstly , ten preconditioning cycles with uniaxial tension - compression up to a load between 10 n and 400 n with a rate of 5 mm / min were applied . then , a monotonic , uniaxial compression was performed at the same rate . the load - displacement curve was recorded and vertebral failure load ( fl ) was defined as the first peak of the load - displacement curve with a subsequent drop of > 10% . all statistical analysis was performed using spss ( spss , chicago , il , usa ) . mean and sd of all mr - based ( fat fraction ) , mdct - based ( bmd and trabecular bone microstructure parameters ) , and biomechanical testing - based ( failure load of each individual vertebra divided by the minimum transverse cross - sectional area of each individual vertebra , i.e. , normalized failure load ) parameters were computed over the ten measured vertebra bodies . the kolmogorov - smirnov test showed for all parameters no significant difference from a normal distribution ( p > 0.05 ) . therefore , correlations between the different parameters were computed with linear regression models and expressed as slope coefficient b , its standard error , correlation coefficient r , and its p value . due to the relatively small sample size in this pilot study , statistical results were expressed as bootstrap derived standard error of the coefficient b and p value of the regression model . figure 1(c ) shows the spectra acquired at different tes on a vertebra , confirming a faster t2 relaxation for the water peak than for the fat peaks and verifying the need for t2 correction to derive a proton density fat fraction . figure 1(d ) shows the experimentally measured spectrum and the fitted spectrum . there is a strong overlap between fat peaks e and f and the water peak , verifying the need for a constrained fitting of peaks e and f to achieve a reliable estimation of the water peak . table 1 summarizes the statistics ( mean and sd values ) of the main measured parameters . the proton density fat fraction ranged from 26% to 43% with a mean value of 32% and a sd of 5% . the transverse cross - sectional area of the vertebra ranged from 4.9 cm to 9.7 cm with a mean value of 6.5 cm and a sd of 1.5 cm . the normalized fl ranged from 157 n / cm to 798 n / cm with a mean value of 442 n / cm and a sd of 251 n / cm . mean and sd values for bmd and trabecular microstructure parameters ( bv / tv , tbn , and fd ) are also listed in table 1 . figure 2(a ) shows a negative correlation of proton density fat fraction with bmd ( r = 0.72 , p = 0.045 ) . figure 2(b ) shows a negative correlation of proton density fat fraction with normalized fl ( r = 0.77 , p = 0.013 ) . proton density fat fraction also correlated with trabecular microstructure parameters , showing a trend close to statistical significance ( p < 0.1 ) for bv / tv , tbn , and fd ( table 2 ) . normalized fl showed strong correlations with bmd and all trabecular microstructure parameters with correlation coefficients ranging between 0.83 and 0.90 with p < 0.01 ( table 2 ) . bootstrap derived standard error of the coefficient b and p value of all regression models validated the obtained results ( table 2 ) . the present in vitro study investigates , using human spine specimens , the relationship between mrs - based vertebral bone marrow proton density fat fraction , mdct - based measures of bmd and trabecular microstructure , and biomechanical strength . a strong correlation was observed between biomechanical strength and bmd and between biomechanical strength and trabecular microstructure parameters . a correlation was also observed between bone marrow proton density fat fraction and biomechanical strength , providing for the first time a direct validation of the negative association between bone strength and bone marrow fat content . the relationship between bone marrow fat content and bmd has been investigated in multiple previous studies [ 7 , 1421 ] . specifically , previous works have shown statistically significant differences in bone marrow fat content between controls and subjects with osteopenia and osteoporosis ( grouped based on their dxa - based t - score ) [ 1618 ] . it has been also shown that there is a negative correlation between fat content and dxa - based bmd [ 7 , 15 ] . the limitation of dxa - based bmd measurements is that they include contributions from both trabecular and cortical bone components . it has not been until recently that trabecular bmd values based on quantitative computed tomography have been used to study the relationship between trabecular bone density and bone marrow fat content [ 16 , 20 ] . the present study used thus mdct - based trabecular bmd measures excluding the contributions from cortical bone compartments in the study between bmd and bone marrow fat content . in vivo measurement of vertebral trabecular microstructure parameters using mdct would not be justifiable in clinical routine due to the need for high spatial resolution and the associated dose limitations ( effective dose of estimated 3 msv according to graeff et al . ) . that is why the acquisition of mdct data ex vivo in specimens constitutes a meaningful step in establishing any association between trabecular microstructure parameters and bone marrow fat content . the negative correlation between bone marrow fat content and bone density has been shown in multiple recent studies [ 7 , 1420 ] . different mechanisms have been proposed for explaining this negative association , suggesting that bone marrow fat is not simply a filler of the bone matrix cavities . proposed mechanisms include a drift in mesenchymal stem cell differentiation that favors adipogenesis over osteoblastogenesis or a direct effect of adipocytes on suppressing osteoblastogenesis . in parallel , it is well known that bone density is positively correlated with biomechanical strength . the present work shows a negative association between marrow fat content and biomechanical strength and provides to the best of our knowledge the first direct validation of the association between bone marrow fat content and biomechanical strength . therefore , the present results complement the existing knowledge about the importance of bone marrow adiposity in understanding the pathophysiology of bone weakening and about the value of the mrs - based proton density fat fraction of bone marrow as an additional useful parameter in monitoring osteoporosis diagnosis , progression , and therapy . however , future larger scale studies would be necessary to understand whether marrow fat content can become a predictor of bone strength after correcting for bmd effects . the present study uses single - voxel mr spectroscopy to measure the bone marrow proton density fat fraction . acquisitions with multiple tes are performed to account for t2 effects and a constrained fitting approach is adopted taking into consideration the bone marrow triglyceride structure to avoid the inclusion of fat peaks e and f in the water peak signal . the consideration of t2 effects and of the presence of the secondary fat peaks ( e and f ) overlapping with water peak aims for the extraction of a proton density fat fraction of bone marrow instead of a signal - weighted fat fraction reported in previous works [ 1618 ] . by using this mr spectra analysis method and an appropriate parameter selection ( long tr to remove any t1 effects and short tes to reduce j - coupling effects ) , mrs using a stimulated echo acquisition mode ( steam ) as preformed in our present study can be successfully applied for in vivo bone marrow fat quantification as recently shown in the proximal femur and spine [ 28 , 34 ] . single - voxel mrs has been the technique most frequently used to measure fat content in different bone marrow regions , including the vertebral bodies and the proximal femur . however , single - voxel mrs provides very poor spatial resolution , which is an important limitation when applied in bone marrow regions with a spatial heterogeneous distribution of fat content . quantitative water - fat imaging techniques have recently been applied for measuring bone marrow proton density fat fraction with high spatial resolution in good agreement with single - voxel mrs after accounting for appropriate confounding factors [ 28 , 35 , 36 ] . this relatively small sample size might be responsible for not reaching statistical significance ( p < 0.1 but p > 0.05 ) when studying the relationship between bone marrow fat content and certain trabecular microstructure parameters ( tbn and fd ) . second , given the limited spatial resolution of mdct , the mdct - based measured values of the trabecular microstructure can not depict the true trabecular structure . however , it has been previously shown that histomorphometric measurements as assessed with mdct and micro - ct correlate significantly . it has been also recently shown that the correlations of failure load versus trabecular bone microstructure parameters obtained with mdct and high - resolution peripheral quantitative computed tomography ( hr - pqct ) are not significantly different . third , the imaging - derived measurements are limited in the prediction of bone strength of an intact vertebra , since they do not account for the endplates , cortical shell , and posterior elements . in conclusion , a negative relationship was observed between bone marrow proton density fat fraction and trabecular bmd and a negative relationship was observed between bone marrow proton density fat fraction and biomechanical strength . this in vitro study confirms the previously reported negative association between bone marrow fat content and bone density and provides the first direct ex vivo validation of a negative association between bone marrow fat content and bone strength . these findings underline the importance of the bone marrow in the pathophysiology and imaging diagnostics of osteoporosis . a 62-year - old man developed sudden dysarthria and weakness of the right upper extremity . on admission , he was alert with intact orientation . the neurological examination revealed an impaired ability to perform rapidly alternating movements in the right upper extremity , but there was no other motor , sensory , or oculomotor abnormality . brain mri including diffusion - weighted imaging revealed an isolated lesion involving the left ventroposteromedial ( vpm ) thalamic nucleus ( fig . gustatory function was assessed using the standardized taste - strips test , and the olfactory function was tested using the korean version of the " sniffin ' sticks " test ii ( kvsst ) . the patient was unable to differentiate sweet , sour , salty , and bitter tastes on both sides of the tongue , and perceived all food as tasteless . his food intake stopped immediately after the stroke , but subsequently gradually returned to normal . kvsst performed 6 months after the thalamic stroke confirmed that gustatory dysfunction was still present in the patient . f - fdg pet imaging ( advance pet scanner , ge , milwaukee , wi , usa ) data obtained from the patient and 12 age - matched normal controls was subjected to spm analysis ( ultra - sparc 10 workstation , sun microsystems , silicon valley , ca , usa ; spm99 , institute of neurology , university college london , uk ) . all reconstructed pet images were spatially normalized into the mni template ( montreal neurological institute , mcgill university , canada ) using the affine transformation of the spm99 program . the normalized images were smoothed by convolution with an isotropic gaussian kernel with an fwhm of 8 mm to increase the signal - to - noise ratio . the images obtained from the patient were statistically compared with those of the 12 healthy volunteers on a voxel by - voxel basis using the t - test to detect significant decreases in the regional metabolism . voxel thresholding at p=0.01 ( uncorrected ) and p=0.001 ( uncorrected ) was used . spm analysis of brain pet images revealed hypometabolism in the left insular cortex ( fig . the solitary tract nucleus in the medulla is the first relay in taste sensation , which subsequently involves the central tegmental tract to the medial aspect of the vpm nucleus , immediately adjacent to the somatosensory area for the oral cavity and fingers . taste information is then relayed to the primary taste cortex in the frontal operculum and insular cortex , finally reaching the secondary cortical taste area in the orbitofrontal cortex.1 from an examination of 15 cases of unilateral gustatory disturbance due to central lesions , onoda and ikeda suggested that the central gustatory pathways in humans ascend ipsilaterally from the solitary nucleus of the medulla oblongata to the pons , from where they cross to a higher position in the midbrain and reach the thalamus contralaterally.2 another clinical and mri investigation also suggested that the central taste pathway in humans projects ipsilaterally from the solitary nucleus up to the level of the upper pontine or lower midbrain before crossing.3 it is generally accepted that the taste pathway projects from the ipsilateral solitary nucleus in the medulla , and immediately crosses and traverses the contralateral medial lemniscus on its way to the thalamus . however , the cortical representation of gustatory fibers in the central pathway from the vpm thalamic nuclei in humans has not been elucidated . this is due to the paucity of functional imaging studies on taste disorders in patients with cortical or thalamic stroke , despite the insular cortex and thalamus having an important role in gustatory and lingual somatosensory function . recent investigations in stroke patients have indicated the presence of ageusia contralateral to a thalamic or corona radiata infarction , supporting that the gustatory fibers ascend contralaterally in the cerebral hemisphere and that the pathway ascends from the thalamus to the cerebral cortex via the posterior part of the corona radiata.4 functional imaging of the central gustatory pathway from the thalamus to the cortex has been mainly performed in nonhuman animals . an anterograde and retrograde tract - tracing study of the projections from the thalamic gustatory area in the rat showed that parvicellular vpm neurons projecting to the amygdaloid complex differed from those projecting to the insular cortex.5 however , recent developments in functional brain mapping techniques using pet have revealed the specific structures in the human brain involved in taste sensation . a pet investigation of taste sensation in healthy humans showed that the thalamus , insular cortex , anterior cingulated gyrus , parahippocampal gyrus , lingual gyrus , caudate nucleus , and temporal gyri participated in the discrimination of a salty taste.6 however , these studies have not elucidated the termination laterality of fibers from the thalamic gustatory relay . the present report is one of only a few on bilateral taste loss associated with acute left unilateral thalamic infarction . moreover , there have been few functional imaging studies on taste disorders in patients with a localized vpm thalamic infarct , which would facilitate identification of the connection between cortical and subcortical areas . we had expected our pet study to reveal bilateral cortical hypometabolism , and hence we can not explain the exact reason for unilateral left insular hypometabolism manifesting as a bilateral taste loss . we postulate that the laterality of taste dominance is in the left hemisphere , with the right hemisphere only involved in the taste sensation of the right hemitongue . hypometabolism in the left thalamus was evident from our pet investigation but not from the spm analysis . we do not know the exact reason for this apparent discrepancy , but it might be attributable to the lesion being too small , even though there were definitely structural and functional impairments in that region . we believe that this case report provides useful information about the laterality of taste and the functional asymmetry of the cerebral hemisphere . further gustatory functional imaging studies are necessary in acute stroke patients to elucidate the cortical representation and laterality of taste .

bone marrow adiposity has recently gained attention due to its association with bone loss pathophysiology . in this study , ten vertebrae were harvested from fresh human cadavers . trabecular bmd and microstructure parameters were extracted from mdct . bone marrow fat fractions were determined using single - voxel mrs . failure load ( fl ) values were assessed by destructive biomechanical testing . significant correlations ( p < 0.05 ) were observed between mrs - based fat fraction and mdct - based parameters ( up to r = 0.72 ) and mrs - based fat fraction and fl ( r = 0.77 ) . these findings underline the importance of the bone marrow in the pathophysiology and imaging diagnostics of osteoporosis . unilateral taste loss is usually observed on the side contralateral to a thalamic infarction , despite gustatory function being represented bilaterally . we report a rare case of bilateral taste loss in a patient with an acute left unilateral thalamic infarction , with unilateral left insular hypometabolism demonstrated by statistical parametric map analysis of pet images . our observations suggest that the left insular cortex and left ventroposteromedial thalamic nuclei are critical to bilateral gustatory sensation .

the aids epidemic which has hit southern africa with such devastating force affects a variety of reproductive issues among infected people . these include whether or not to become pregnant , when and which type of contraception to use , and decisions on whether to continue or try to terminate a pregnancy . findings from studies in both developing and developed nations indicate that a significant proportion of hiv infected adults still desire or intend to have children [ 14 ] . with the advent of highly active antiretroviral therapy ( haart ) and its impact on the health of the infected as well as its impact on lowering mother to child transmission of hiv , the number of hiv infected people considering childbearing has increased [ 57 ] . while much attention has been paid to the reproductive choices and intentions of the infected as well as to some factors that influence these [ 14 ] , most studies are silent on the role of health professionals in the reproductive and sexual lives of hiv infected people . there are few studies that mention the role of health professionals in the reproductive sphere of hiv positive people , yet these are the people who usually play a significant role in the decision making process of hiv positive people . this is because of their medical expertise and that most hiv infected people make their reproductive decisions within a biomedical and health institution context [ 7 , 9 , 10 ] . there are some studies that have involved health professionals and they found that biomedical considerations usually dominate health care providers ' attitudes towards reproduction by hiv infected people [ 11 , 12 ] . findings from this study indicate that there are three dominant discourses concerning childbearing by people with hiv among health professionals . the first two are rooted in the biomedical model of health where biomedical considerations are prominent while the third takes a human rights perspective . taped in - depth interviews were conducted with 12 health professionals in two opportunistic infections clinics in bulawayo . health professional interviewees were selected for their involvement in the care , treatment , and counselling of hiv positive people . this research among health professionals was part of a broader study that explored reproductive decisions among hiv positive couples in bulawayo , zimbabwe , in 2010 . interviews with hiv positive couples revealed that people who featured most in their reproductive and sexual lives were the health professionals ( hps ) ( nurses , doctors , and counsellors ) in opportunistic infections clinics and support groups . as a result the defining characteristic for this sample was involvement with hiv positive people in the past two years . using these characteristics , six counsellors , four nurses , and two doctors were interviewed by the researcher at their place of work . informed consent was obtained from all the respondents and so was the right to tape record the interviews . more counsellors than any other group of hps were selected because , according to the interviews with hiv positive people , they seemed to interact with them more than the nurses or the doctors . data were analysed using a grounded theory approach , based on a process that helps researchers to discover categories , themes , and patterns that emerge from the data . transcribed interviews were then content analysed to identify patterns and themes concerning attitudes of health professionals towards reproduction by hiv positive couples . the strategies used in the data analysis were a systematic review and a thoughtful reading of interview data , coding , categorising , and sorting for patterns , and the construction of the story told . this study found that the majority of hps interviewed generally gave people incomplete information on reproduction . the information given to hiv positive couples centred on pregnancy prevention and prevention of reinfection . the possibility of having children where and when it was discussed was usually presented as a dangerous possibility that people must try to avoid . responding to the question on what they tell people with hiv regarding reproduction some respondents had this to say;we tell them there is re - infection when you have intercourse with an infected person so the first piece of information that we give them is the proper use of the condom ( male counsellor).the use of the condom reduces the re - infection rate and apart from that it prevents pregnancy . if you get pregnant there are high chances of getting an hiv positive child ( nurse).what we tell them is that its not wise to be pregnant when one is hiv positive ( nurse ) . we tell them there is re - infection when you have intercourse with an infected person so the first piece of information that we give them is the proper use of the condom ( male counsellor ) . the use of the condom reduces the re - infection rate and apart from that it prevents pregnancy . if you get pregnant there are high chances of getting an hiv positive child ( nurse ) . what we tell them is that its not wise to be pregnant when one is hiv positive ( nurse ) . another counsellor also pointed out that they emphasised what he called practical aspects when giving hiv positive people information on their future reproductive prospects . he said they emphasised the issue of reinfection and drug resistance if people do not use condoms and the negative repercussions this has not only for them but also for the opportunistic infections clinics and others who are hiv positive . the practicalities that they are made aware of are in a way meant to steer them from the path of unsafe sex and dissuade them from having children . the manner in which the said objective information regarding reproduction was delivered reveals the subjective prejudices of most hps interviewed . all of the counsellors and some nurses indicated that they had in the past months ( before july 2010 ) been approached by an hiv positive individual or couple who wanted to have a child . most hps empathised and some sympathised with them and were aware of the social and personal challenges faced by these people . one counsellor said because you are young , you are a newly we d couple , you meet a lot of challenges and in an african situation where you are a daughter in law , you can not be a daughter in - law without children . because you are young , you are a newly we d couple , you meet a lot of challenges and in an african situation where you are a daughter in law , you can not be a daughter in - law without children . though aware of the sociocultural constraints faced by most of these couples or individuals , most health professionals still felt that they had to seek medical approval before they could reproduce . some pointed out that these people could have a child provided they fulfilled certain conditions set by the health professionals ( conditional choice stance ) . others were of the view that though their need to have a child may be genuine it was not necessary to have a child considering their condition ( antichildbearing stance ) . they felt the risks posed to both the mother and the child far outweighed the need to have a child . examining the information given to those who desire children , one can not help but notice that the information itself and the manner in which it was given were , in counselling principles , generally biased and meant to discourage childbearing . responding to how he would deal with someone desiring to have a child , one counsellor responded this way;well , our most important area of discussion is ; it is still possible to be pregnant and get a child when you are hiv positive but then our area of interest is what does it mean to be pregnant when you are hiv positive it means whilst we had built your health so much with arvs the stress and strain related to delivery and the psychological pressure related to nursing a child may actually be counter to what the arvs are trying to achieve . well , our most important area of discussion is ; it is still possible to be pregnant and get a child when you are hiv positive but then our area of interest is what does it mean to be pregnant when you are hiv positive it means whilst we had built your health so much with arvs the stress and strain related to delivery and the psychological pressure related to nursing a child may actually be counter to what the arvs are trying to achieve . other counsellors said there is a lot that goes on when you are pregnant . pregnancy lowers your immunity and you are then open to other infections , these other infections will also lower your cd4 cell count and increase the rate of progression to aids . you feel pity for them , really it 's a young couple , they have no child , they really want to have a child but just because of this ( hiv ) they can not . there is a lot that goes on when you are pregnant . pregnancy lowers your immunity and you are then open to other infections , these other infections will also lower your cd4 cell count and increase the rate of progression to aids . you feel pity for them , really it 's a young couple , they have no child , they really want to have a child but just because of this ( hiv ) they can not . most hps interviewed emphasised the negative aspects of childbearing when giving potential parents advice . apart from the fact that the validity of this information can be disputed , the manner in which it was delivered made it seem compulsory for hiv positive people to act upon . this deviates from the professional expectation from counsellors to give clients objective , unbiased , and unprejudiced information . the study found that there are three dominant discourses among hps concerning reproduction by hiv positive couples or people . these are the conditional choice discourse , the antichildbearing discourse , and the prorights discourse . the study found that six of the hps interviewed took a conditional choice stance , while four took an antichildbearing stance and only two took a prorights stance . the conditional choice stance argues that hiv positive people may have children as long as they made their decision on the basis of information and advice given to them by hps . there are a number of conditions that these health professionals indicate as necessary to be fulfilled before hiv positive people can have children . it is important to note that these conditions are to be determined by the hps though on the other hand they claim to be neutral facilitators . the following are some of the conditions pointed out:they have to do that ( decision making ) on an informed basis.i think people need information , the correct information and be allowed to make choices based on correct information.people can have children as long as they are able to make sure that they make every effort that child does not become infected.if they want to make a decision to get pregnant they have to consult a doctor who will advise them on how big the risk of getting pregnant is.they have to consult a medical person who will look at their cd4 cell counts , how they are clinically and what risk there is.we also check the stage ( of aids ) they are in.i referred them to their private doctor so he may tell them whether they can have a child or not . they have to do that ( decision making ) on an informed basis . i think people need information , the correct information and be allowed to make choices based on correct information . people can have children as long as they are able to make sure that they make every effort that child does not become infected . if they want to make a decision to get pregnant they have to consult a doctor who will advise them on how big the risk of getting pregnant is . they have to consult a medical person who will look at their cd4 cell counts , how they are clinically and what risk there is . we also check the stage ( of aids ) they are in . i referred them to their private doctor so he may tell them whether they can have a child or not . it seems as far as these hps are concerned hiv positive people have to fulfil and adhere to certain conditions determined by the health professionals before they can think of having children . the proponents of this stance regard childbearing by people living with hiv as an unnecessary risk to the unborn child . as far as they are concerned it is not only irrational but also immoral for hiv positive people to have children . it is immoral because there is a possibility of having a positive child and thus causing suffering to an innocent soul the hps who take this stance in a way argue that if it is irrational to allow the worst outcome of our actions , and if it is immoral to cause suffering , then it is irrational and immoral for hiv positive people to have children . are some of their responses regarding childbearing among those who are infected: .. this is a problem ( having children ) . the painful thing is to see the children when they get sick of aids that is painful especially if you deal with children who are hiv positive which is what i do most of the time ( counsellor).i do not think it is necessary ( to have a child)it 's not necessary . i believe there can still be a happy marriage without children and perhaps my opinion is heavily influenced by my medical background . i wish i could come out of it and stand on neutral ground , but i do not think it 's necessary , they should not ( doctor). both of you , you are ill now and the child will be ill as well and the child will be in and out of hospital now and again or you have a child and five years down the line both of you die what will happen to the child and worse if the child is positive as well , even if its negative what happens to her , and so forth they are very innocent but they are suffering ( nurse ) . .. this is a problem ( having children ) . the painful thing is to see the children when they get sick of aids that is painful especially if you deal with children who are hiv positive which is what i do most of the time ( i do not think it is necessary ( to have a child)it 's not necessary . i believe there can still be a happy marriage without children and perhaps my opinion is heavily influenced by my medical background . i wish i could come out of it and stand on neutral ground , but i do not think it 's necessary , they should not ( both of you , you are ill now and the child will be ill as well and the child will be in and out of hospital now and again or you have a child and five years down the line both of you die what will happen to the child and worse if the child is positive as well , even if its negative what happens to her , and so forth the response of this group to pregnancy among women on treatment or to couples / individuals who intend to reproduce is usually anger , disappointment , dejection , and a feeling of defeat and failure . its advocates argued that it is the right of every human being to choose freely without fear or fetters . it is also everyone 's right to be given the correct and complete information regarding reproduction when they need it . they argued thatevery human being has a right , has every right to decide what he wants concerning his health , family , just about everything . as we have here at oi clinics there are people who choose not to take arvs , that is the choice of the individual , it is his right every human being has a right , has every right to decide what he wants concerning his health , family , just about everything . as we have here at oi clinics there are people who choose not to take arvs , that is the choice of the individual , it is his right another counsellor argued that due to the number of adults infected with hiv in zimbabwe and given the high chances of having a negative child people have the right to choose to reproduce freely . he saidit is very important , to me i think it is very important for hiv positive people to have children because even if i give you our statistics in zimbabwe , it says around 20% of the adult population are people living with hiv . so considering the chances of these hiv positive people having negative children if they take the necessary drugs i think there is no reason for them not to have children . so i think being hiv positive should not ever be used to stop someone from having children . it is very important , to me i think it is very important for hiv positive people to have children because even if i give you our statistics in zimbabwe , it says around 20% of the adult population are people living with hiv . so considering the chances of these hiv positive people having negative children if they take the necessary drugs i think there is no reason for them not to have children . so i think being hiv positive should not ever be used to stop someone from having children . study findings revealed that there are three dominant discourses on the issue of childbearing by hiv positive people among health professionals . prevalent in the conditional choice and antichildbearing discourses is the view of hiv positive people as abnormals who do not have the same reproductive rights and freedom as normals . from these perspectives hiv / aids is framed as a disability and like most people with disabilities hiv positive people find themselves in a position where their condition is viewed as a handicap . persons with disabilities often face serious discrimination based on attitudes , perceptions , misunderstandings , and lack of awareness about disability [ 15 , 16 ] . similarly people with hiv / aids who desire to reproduce usually face discrimination as a result of negative social constructs of hiv / aids . as asch and fine ( 1988 ) note , the attitudes and structural barriers of the nondisabled turn disabilities into handicaps . ferri and gregg ( 1998 ) argue that the reasoning behind such a stereotype is the fear that people with disabilities will produce defective offspring . in the case of hiv positive people the fear seems to be not only that they will produce infected children but also that they will increase the number of orphans and child headed households in the society due to their early death . as a result people with hiv / aids find their right to reproduce being questioned and sometimes denied by both the medical fraternity and the society . there seems to be a view in this school of thought that the information given to hiv positive people should make them arrive at what is seen as an ideal decision , that is , not to procreate . such an attitude may discourage hiv positive people from consulting hps on issues relating to their sexual and reproductive health and life and this may consequently lead to negative health outcomes for hiv positive people . the prorights stance argues that like any normals , hiv positive people have the right to do what they want , when they want , in the matter of reproduction . this perspective does not frame hiv / aids as a disabling factor but rather a chronic but manageable condition . however the consideration of hiv positive people as abnormals in the society in general and in the medical fraternity in particular means that hps with such a liberal view are likely to be few . as a result their influence on the reproductive lives of people with hiv / aids is also likely to be limited . there is also a tendency among most hps to overemphasise the risk to the child though they are aware that this risk is considerably reduced with the help of prophylactic drugs . the way the risk to the child and the mother is emphasised makes it loom larger in the minds of hiv positive people than it actually is given the fact that hps are considered as authorities in this field by hiv positive people . there was a tendency among hps to omit information on strategies to reduce reinfection as well as mtct when giving hiv positive people information regarding reproduction . the information on how they can lower the rate of reinfection and the incidence of mtct and thus increase their chances of getting an hiv negative child was usually not given prominence . in their accounts of the reproductive information given to people , ten of the twelve hps interviewed were silent on the role of nevirapine and other arvs in lowering the incidence of mtct and how those who want children can take advantage of this . when probed on their silence on arvs one counsellor quipped , true as this may be , hiv positive people deserve to know all the possibilities available to them and it is the duty of hps to avail that information to them . health professionals ' attitudes towards childbearing among hiv infected couples were largely shaped by their medical background . the issue of reproduction tended to be medicalised and the aspects of patients ' lives and experiences that transcended the biomedical milieu tended to be neglected [ 11 , 19 ] . health professionals in the study were more concerned about the potential adverse effects of pregnancy , reinfection , and drug resistance than with the personal and social meaning of childbearing to their patients or their reproductive rights . this tendency to lean towards the biomedical model of health which by its nature is concerned more about the physical rather than the emotional and social health meant that there was a tendency among most health professionals to prescribe rather than advise a course of action . many health professionals in the study felt that deciding to have a child required careful planning and consideration , with a right time to fall pregnant , which included an adequately high cd4 count , access to art and pptct programmes , and whether the individual was physically healthy . right time was supposed to be determined by them or by some other health professionals . while the physical health of the infected is important , their psychosocial needs were usually ignored by this dominant discourse . the psychosocial aspect of health is as important as the physical in the overall health of the individual . the study results indicate that the stance of most hps in the area of reproduction among people with hiv is that of interested parties who instead of giving value free information give value laden , authoritative advice . the information given to hiv positive people generally emphasises safe sex and the risks involved in pregnancy while discounting the low risk of having a positive child when one is on haart . recent studies have indicated that mtct risk is very low among women on haart and takes nevirapine at the onset of labour [ 5 , 6 ] . there seems to be a general tendency among the hps to commit the error of omission when giving hiv positive people reproductive information . these are the principles of medical ethics , the oi clinic 's criteria on arv treatment , the biomedical underpinning of the field of medicine , and to a certain extent the personal views of hps on reproduction by hiv positive people . the biomedical model of health is by its nature prescriptive and it views the hp as the authority in terms of hp - patient relationship . modern medicine , argues samson ( 1999 ) , is based on a mechanistic , materialist view of the body and the hp , as the professional , exercises control over this body . the unequal relationship between health professionals and their patients where the doctor is in a position to determine how a health problem should be dealt with and the patient is not has come to be accepted as people normally do not talk back to the hps ; they just listen passively . as one nurse pointed out during the research,people generally have an impression that a nurse is someone who would just instruct you to do this and that . now we have a challenge to change the whole process so that people can be able to approach us freely . people generally have an impression that a nurse is someone who would just instruct you to do this and that . now we have a challenge to change the whole process so that people can be able to approach us freely . the interaction between hps and hiv positive people is embedded in a social and political context in which hps have medical knowledge and technical expertise that patients usually lack . as a result of their position as gatekeepers and because of the power they exercise over the behaviour of their patients they may feel obliged to prescribe rather than to advise their patients . the biomedical perspective taken by most hps in this research may also be based on a genuine concern for the health and wellbeing of hiv positive people as well as for children born to them and a misguided view that curtailing the reproductive rights of hiv positive people is ethical . until recently , hiv / aids has been a fatal disease and still is a potentially lethal and infectious condition . thus the concern and involvement of hps in the reproductive decision making process of hiv positive people may emanate from a rational logic to safeguard the health of hiv positive people . nonetheless , however altruistic their aims may be , the deliberate omission of important information relating to childbearing displayed by some hps in the study is not medically or ethically justifiable . as noted above this study was conducted in two urban opportunistic infections clinics that were fairly well resourced . as a result of the specificity of this study it possibly may not be representative of the views and attitudes of the majority of health professionals in other urban , nonurban , or resource poor settings . the results of this study indicate a general negativity towards reproduction by hiv positive people among health professionals . most of the reproductive information and advice given to hiv positive people was geared towards discouraging them from childbearing . the information was neither unbiased nor complete and the advice given to hiv positive people was not undirective or neutral as is required of counsellors . the study results also reveal a lack of clear guidelines in dealing with the issue of reproduction among people with hiv and hence the existence of varying discourses on the issue and the haphazard advice given to the patients by different hps . though health professionals are entitled to their own ideologies there is a need for clear counselling guidelines for hps on how to deal with hiv positive people who need to reproduce so as to harmonise the advice given to them . this need is urgent as the number of hiv positive people who intend to reproduce is likely to increase as more people are admitted into the highly active antiretroviral therapy program . according to a single - blind , within - subject comparison , 15 healthy , normal - weight men ( mean age se , 28.5 1.0 years ; bmi , 23.1 0.5 kg / m ) participated in two hyperinsulinemic - euglycemic clamp experiments ( human insulin , detemir ) spaced apart at least 1 week . all subjects gave written informed consent to the experiments , which were approved by the local ethics committee . in both experimental conditions , an insulin bolus injection ( 17.75 mu / kg body wt [ = 0.1065 nmol / kg body wt ] human insulin , insulin actrapid ; novo nordisk , bagsvrd , denmark ; 90 mu / kg body wt [ = 2.16 nmol / kg body wt ] detemir , insulin levemir ; novo nordisk ) was given followed by a steady 90-min infusion of 1.0 mu kg min (= 0.006 nmol kg min ) human insulin versus 2.0 mu kg min (= 0.048 nmol kg min ) detemir . to compensate for the relatively slower onset of the action of detemir , a higher dosage was chosen ( 15,17 ) to induce comparable peripheral effects of both compounds as assessed by rates of glucose infusion necessary to keep blood glucose levels in the euglycemic range . volunteers reported to the laboratory at 0800 , after an overnight fast of 10 h , and were prepared for electroencephalographic recordings , insulin infusions , and blood samplings . we inserted venous cannulas into the subject 's arms and connected them to tubes enabling infusion and blood sampling from an adjacent room without awareness of the subject . the arm from which samples were taken was positioned in a heated box ( 55c ) to enable drawing of arterialized venous blood . during recordings , subjects sat in a reclining chair in a sound - attenuated room of constant temperature , with their heads stabilized by a cushion . they were instructed to relax and not to move during recordings and to fixate their gaze on the wall in front of them . subjects pressed a button every estimated 30 s to maintain a constant state of mental activity and to not doze off . recordings of dc potentials started at 0940 with a baseline phase of 20 min followed by the bolus injection of human insulin and detemir , respectively ( t = 0 ) . subsequent insulin infusion lasted for 90 min , ending at 1130 when eeg recordings were also stopped . arterialized blood was drawn at 15-min intervals during baseline , at 5-min intervals during insulin infusion , and at 15-min intervals thereafter to monitor blood glucose concentration ( hemocue b - glucose - analyzer , hemocue ab , angelholm , sweden ) . during and after insulin administration , subjects intravenously received a 20% glucose solution at a variable rate to maintain normal plasma glucose levels . blood samples for the determination of hormonal parameters were repeatedly collected , and routine assays were used to determine concentrations of serum c - peptide , plasma acth , serum cortisol ( all immulite ; dpc , los angeles , ca ) , plasma glucagon ( ria ; adaltis , montreal , quebec , canada ) , and serum leptin ( ria ; linco research , st . charles , mo ) . at 1150 ( i.e. , 20 min after insulin infusion had stopped ) , a standardized buffet of around 4,650 kcal was offered from which subjects were allowed to eat ad libitum during the subsequent 50 min ( table 1 ) . subjects were kept unaware of hypothesized treatment effects on food intake and were not aware that their food intake was measured by weighing buffet components before and after food intake . in addition , to prevent overeating , subjects were allowed to take with them any remaining food afterward . before ( at 0915 ) and after ( at 1135 ) recordings and at the end of the session at 1300 , subjects rated their hunger , thirst , and tiredness on 10-point scales and completed a questionnaire assessing alertness and autonomic symptoms on 5-point bipolar scales of 20 contrasting adjective pairs ( e.g. , activated - inert and sweating - shivery ) ( 22 ) . they also filled in a checklist of 161 adjectives assessing mood on 14 dimensions ( 23 ) . composition of the test buffet composition of the buffet offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . standard recordings of dc potentials , electro - oculogram , and electromyogram were performed as described previously ( 24,25 ) . dc - potential recordings were obtained from left and right frontal ( f3 , f4 ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) electrodes referenced to linked electrodes at the mastoids . a brainamp dc amplifier ( brain vision , london , u.k . ; low - pass filter : 30 hz , sampling rate : 200 hz ) was used . dc - potential drifts with short - circuited input were constantly < 5 v / h , and electrode impedance , measured before and after recordings , never exceeded 5 k. average dc - potential values were determined offline for subsequent 5-s intervals . linear potential drifts during the 20-min baseline period extending into the 90-min insulin infusion period were removed using a linear regression method . periods where electromyogram or electro - oculogram indicated increased muscular activity or eye movements were excluded from analysis . the average dc potential during baseline was set to 0 v , and potential shifts during treatment were expressed as difference values . differences in dc - potential values between conditions were evaluated first on an exploratory basis by point - wise comparisons using t tests to identify time ranges with most consistent differences ( 26 ) . the time range selected for analysis covered 2190 min of insulin infusion . values for this time interval were then subjected to anova , including the repeated - measures factors treatment ( human insulin vs. detemir ) and topography ( electrode locations ) . post hoc contrasts were used to specify significant anova main effects and interactions . for the dc - potential analysis , data from four subjects had to be excluded because of technical failures and artifacts of apparent nonbiological origin . behavioral measures and hormonal parameters were analyzed with anova and paired t tests as appropriate . according to a single - blind , within - subject comparison , 15 healthy , normal - weight men ( mean age se , 28.5 1.0 years ; bmi , 23.1 0.5 kg / m ) participated in two hyperinsulinemic - euglycemic clamp experiments ( human insulin , detemir ) spaced apart at least 1 week . all subjects gave written informed consent to the experiments , which were approved by the local ethics committee . in both experimental conditions , an insulin bolus injection ( 17.75 mu / kg body wt [ = 0.1065 nmol / kg body wt ] human insulin , insulin actrapid ; novo nordisk , bagsvrd , denmark ; 90 mu / kg body wt [ = 2.16 nmol / kg body wt ] detemir , insulin levemir ; novo nordisk ) was given followed by a steady 90-min infusion of 1.0 mu kg min (= 0.006 nmol kg min ) human insulin versus 2.0 mu kg min (= 0.048 nmol kg min ) detemir . to compensate for the relatively slower onset of the action of detemir , a higher dosage was chosen ( 15,17 ) to induce comparable peripheral effects of both compounds as assessed by rates of glucose infusion necessary to keep blood glucose levels in the euglycemic range . volunteers reported to the laboratory at 0800 , after an overnight fast of 10 h , and were prepared for electroencephalographic recordings , insulin infusions , and blood samplings . we inserted venous cannulas into the subject 's arms and connected them to tubes enabling infusion and blood sampling from an adjacent room without awareness of the subject . the arm from which samples were taken was positioned in a heated box ( 55c ) to enable drawing of arterialized venous blood . during recordings , subjects sat in a reclining chair in a sound - attenuated room of constant temperature , with their heads stabilized by a cushion . they were instructed to relax and not to move during recordings and to fixate their gaze on the wall in front of them . subjects pressed a button every estimated 30 s to maintain a constant state of mental activity and to not doze off . recordings of dc potentials started at 0940 with a baseline phase of 20 min followed by the bolus injection of human insulin and detemir , respectively ( t = 0 ) . subsequent insulin infusion lasted for 90 min , ending at 1130 when eeg recordings were also stopped . arterialized blood was drawn at 15-min intervals during baseline , at 5-min intervals during insulin infusion , and at 15-min intervals thereafter to monitor blood glucose concentration ( hemocue b - glucose - analyzer , hemocue ab , angelholm , sweden ) . during and after insulin administration , subjects intravenously received a 20% glucose solution at a variable rate to maintain normal plasma glucose levels . blood samples for the determination of hormonal parameters were repeatedly collected , and routine assays were used to determine concentrations of serum c - peptide , plasma acth , serum cortisol ( all immulite ; dpc , los angeles , ca ) , plasma glucagon ( ria ; adaltis , montreal , quebec , canada ) , and serum leptin ( ria ; linco research , st . at 1150 ( i.e. , 20 min after insulin infusion had stopped ) , a standardized buffet of around 4,650 kcal was offered from which subjects were allowed to eat ad libitum during the subsequent 50 min ( table 1 ) . subjects were kept unaware of hypothesized treatment effects on food intake and were not aware that their food intake was measured by weighing buffet components before and after food intake . in addition , to prevent overeating , subjects were allowed to take with them any remaining food afterward . before ( at 0915 ) and after ( at 1135 ) recordings and at the end of the session at 1300 , subjects rated their hunger , thirst , and tiredness on 10-point scales and completed a questionnaire assessing alertness and autonomic symptoms on 5-point bipolar scales of 20 contrasting adjective pairs ( e.g. , activated - inert and sweating - shivery ) ( 22 ) . they also filled in a checklist of 161 adjectives assessing mood on 14 dimensions ( 23 ) . composition of the test buffet composition of the buffet offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . standard recordings of dc potentials , electro - oculogram , and electromyogram were performed as described previously ( 24,25 ) . dc - potential recordings were obtained from left and right frontal ( f3 , f4 ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) electrodes referenced to linked electrodes at the mastoids . a brainamp dc amplifier ( brain vision , london , u.k . ; low - pass filter : 30 hz , sampling rate : 200 hz ) was used . dc - potential drifts with short - circuited input were constantly < 5 v / h , and electrode impedance , measured before and after recordings , never exceeded 5 k. average dc - potential values were determined offline for subsequent 5-s intervals . linear potential drifts during the 20-min baseline period extending into the 90-min insulin infusion period were removed using a linear regression method . periods where electromyogram or electro - oculogram indicated increased muscular activity or eye movements were excluded from analysis . the average dc potential during baseline was set to 0 v , and potential shifts during treatment were expressed as difference values . differences in dc - potential values between conditions were evaluated first on an exploratory basis by point - wise comparisons using t tests to identify time ranges with most consistent differences ( 26 ) . the time range selected for analysis covered 2190 min of insulin infusion . values for this time interval were then subjected to anova , including the repeated - measures factors treatment ( human insulin vs. detemir ) and topography ( electrode locations ) . post hoc contrasts were used to specify significant anova main effects and interactions . for the dc - potential analysis , data from four subjects had to be excluded because of technical failures and artifacts of apparent nonbiological origin . behavioral measures and hormonal parameters were analyzed with anova and paired t tests as appropriate . 1a ; p > 0.14 for all comparisons ) , resulting in identical total amounts of energy supplied via glucose infusion ( human insulin , 240.8 23.3 kcal ; detemir , 239.5 25.0 kcal ; p > 0.93 ) . correspondingly , blood glucose concentrations were comparable throughout the experiment ( fig . 1b ; p > 0.34 for all comparisons ) as well as when restricting analysis to the period of insulin infusion ( p > 0.13 ) . serum c - peptide concentrations did not differ between conditions during insulin infusion ( p > 0.25 ) but rose to slightly elevated levels in the human insulin compared with the detemir condition at the end of experiments ( fig . 1d ) as well as leptin did not differ between conditions ( all comparisons , p > 0.40 ) . concentrations of plasma acth ( p > 0.57 ) and serum cortisol ( p > 0.71 ) were likewise similar in both conditions , with cortisol levels showing the expected meal - related increase ( f = 4.19 , p < 0.008 ) . a : rates of glucose infused to maintain euglycemia and concentrations of ( b ) blood glucose , ( c ) serum c - peptide , and ( d ) serum glucagon during intravenous infusion of human insulin ( ) and insulin detemir ( ) , respectively . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . at 20 min after the end of infusions , subjects ate ad libitum from a test buffet . the dc potential in the detemir condition showed a marked negative shift shortly after insulin injection that reached maximum values exceeding 600 v toward the end of the recording epoch ( fig . analyses of average dc - potential levels during the relevant interval from 21 min after the start until the end of insulin infusions confirmed a distinctly more negative potential level in the detemir than human insulin condition ( f = 7.03 , p = 0.02 ; table 2 ) , with this effect displaying an even topographic distribution ( f = 0.59 , p > 0.59 , for treatment electrode location ) . comparisons with preinjection baseline levels confirmed significance for the negative dc potential in the detemir condition during 2190 min of insulin infusion ( f = 8.53 , p = 0.02 ; f = 1.04 , p > 0.39 for electrode location ) . although dc potentials in the human insulin condition appeared to shift slightly toward positive values over central positions , respective analyses did not yield significant differences from baseline values ( f = 0.17 , p > 0.43 ; table 2 ) . average dc potentials recorded from left and right electrodes over frontal ( f3 , f4 , respectively ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) cortical areas before and during intravenous infusion of human insulin ( thin lines ) and insulin detemir ( bold lines ) , respectively . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . in both conditions , rows of asterisks indicate significance ( p < 0.05 ; t tests ) for point - wise comparisons of the potential levels between conditions ( upper row ) and between the detemir condition and respective baseline levels ( lower row ) . dc - potential levels during euglycemic infusion of human insulin and detemir average dc - potential levels ( in v ) over left and right frontal ( f3 , f4 ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) cortical areas from 21 to 90 min of intravenous infusion of human insulin ( hi ) and insulin detemir ( det ) , respectively . the right three columns indicate significance for differences , respectively , between conditions and between the potential levels in the human insulin and detemir conditions and respective baseline levels ( t test ; n= 11 ) . detemir in comparison with human insulin significantly reduced food consumption by 303 135.7 kcal . macronutrient comparisons suggested this effect was particularly pronounced for protein and , to a lesser extent , for carbohydrate intake , but there was no significant statistical interaction between the factors treatment and macronutrients ( f = 1.63 , p > 0.22 ) . the reduction in total energy consumption and also carbohydrate intake by detemir in comparison with human insulin was also observed in analyses taking into account the energy received in the form of intravenous glucose ( table 3 ) . differences in carbohydrate and protein intake between conditions correlated significantly with respective differences in dc - potential shifts ( averaged over all recording sites ) from 21 to 90 min of infusions ( r = 0.74 , p < 0.04 , and r = 0.87 , p < 0.01 , respectively , bivariate pearson coefficients ) . the respective correlation with total energy intake failed to reach significance ( r = 0.61 , p = 0.11 ; r = 0.21 , p > 0.62 for fat intake ) . food intake after euglycemic infusion of human insulin and detemir food intake from a test buffet of 4,650 kcal offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . bottom lines indicate food consumption including the amount of energy infused as glucose to maintain euglycemia until the end of the test buffet . right column indicates significance for differences between conditions ( t test ; n = 15 ) . ten - point scale hunger ratings did not differ between conditions , showing the expected decline from baseline ( detemir , 4.87 0.68 ; human insulin , 5.93 0.54 ; p > 0.11 ) and post - eeg recording values ( 5.87 0.62 vs. 5.93 0.56 , p > 0.91 ) to low postingestion levels ( 1.47 0.43 vs. 1.00 0.24 , p > 0.33 ; p > 0.12 for treatment time ) . a comparable pattern without significant differences between conditions was observed for thirst ( all p > 0.12 ) and tiredness ( p > 0.33 ) ratings . according to the bipolar questionnaire , subjects in the detemir compared with the human insulin condition felt more critical ( critical - comfortable , 2.87 0.13 vs. 3.27 0.18 , p < 0.009 ) and reported increased wakefulness ( sleepy - awake , 3.33 0.23 vs. 2.73 0.23 , p < 0.03 ) and hunger ( full - hungry , 4.20 0.20 vs. 3.53 0.22 , p < 0.01 ) immediately after infusions . the mood adjective checklist did not yield significant differences between conditions for any of the subscales . 1a ; p > 0.14 for all comparisons ) , resulting in identical total amounts of energy supplied via glucose infusion ( human insulin , 240.8 23.3 kcal ; detemir , 239.5 25.0 kcal ; p > 0.93 ) . correspondingly , blood glucose concentrations were comparable throughout the experiment ( fig . 1b ; p > 0.34 for all comparisons ) as well as when restricting analysis to the period of insulin infusion ( p > 0.13 ) . serum c - peptide concentrations did not differ between conditions during insulin infusion ( p > 0.25 ) but rose to slightly elevated levels in the human insulin compared with the detemir condition at the end of experiments ( fig . 1d ) as well as leptin did not differ between conditions ( all comparisons , p > 0.40 ) . concentrations of plasma acth ( p > 0.57 ) and serum cortisol ( p > 0.71 ) were likewise similar in both conditions , with cortisol levels showing the expected meal - related increase ( f = 4.19 , p < 0.008 ) . a : rates of glucose infused to maintain euglycemia and concentrations of ( b ) blood glucose , ( c ) serum c - peptide , and ( d ) serum glucagon during intravenous infusion of human insulin ( ) and insulin detemir ( ) , respectively . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . at 20 min after the end of infusions , subjects ate ad libitum from a test buffet . the dc potential in the detemir condition showed a marked negative shift shortly after insulin injection that reached maximum values exceeding 600 v toward the end of the recording epoch ( fig . analyses of average dc - potential levels during the relevant interval from 21 min after the start until the end of insulin infusions confirmed a distinctly more negative potential level in the detemir than human insulin condition ( f = 7.03 , p = 0.02 ; table 2 ) , with this effect displaying an even topographic distribution ( f = 0.59 , p > 0.59 , for treatment electrode location ) . comparisons with preinjection baseline levels confirmed significance for the negative dc potential in the detemir condition during 2190 min of insulin infusion ( f = 8.53 , p = 0.02 ; f = 1.04 , p > 0.39 for electrode location ) . although dc potentials in the human insulin condition appeared to shift slightly toward positive values over central positions , respective analyses did not yield significant differences from baseline values ( f = 0.17 , p > 0.43 ; table 2 ) . average dc potentials recorded from left and right electrodes over frontal ( f3 , f4 , respectively ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) cortical areas before and during intravenous infusion of human insulin ( thin lines ) and insulin detemir ( bold lines ) , respectively . infusions started with an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg min ) . in both conditions , rows of asterisks indicate significance ( p < 0.05 ; t tests ) for point - wise comparisons of the potential levels between conditions ( upper row ) and between the detemir condition and respective baseline levels ( lower row ) . dc - potential levels during euglycemic infusion of human insulin and detemir average dc - potential levels ( in v ) over left and right frontal ( f3 , f4 ) , frontocentral ( fc3 , fc4 ) , and central ( c3 , c4 ) cortical areas from 21 to 90 min of intravenous infusion of human insulin ( hi ) and insulin detemir ( det ) , respectively . the right three columns indicate significance for differences , respectively , between conditions and between the potential levels in the human insulin and detemir conditions and respective baseline levels ( t test ; n= 11 ) . detemir in comparison with human insulin significantly reduced food consumption by 303 135.7 kcal . macronutrient comparisons suggested this effect was particularly pronounced for protein and , to a lesser extent , for carbohydrate intake , but there was no significant statistical interaction between the factors treatment and macronutrients ( f = 1.63 , p > 0.22 ) . the reduction in total energy consumption and also carbohydrate intake by detemir in comparison with human insulin was also observed in analyses taking into account the energy received in the form of intravenous glucose ( table 3 ) . differences in carbohydrate and protein intake between conditions correlated significantly with respective differences in dc - potential shifts ( averaged over all recording sites ) from 21 to 90 min of infusions ( r = 0.74 , p < 0.04 , and r = 0.87 , p < 0.01 , respectively , bivariate pearson coefficients ) . the respective correlation with total energy intake failed to reach significance ( r = 0.61 , p = 0.11 ; r = 0.21 , p > 0.62 for fat intake ) . food intake after euglycemic infusion of human insulin and detemir food intake from a test buffet of 4,650 kcal offered 20 min after infusion of human insulin and insulin detemir , respectively , had been stopped and from which subjects were allowed to eat ad libitum for 50 min . bottom lines indicate food consumption including the amount of energy infused as glucose to maintain euglycemia until the end of the test buffet . right column indicates significance for differences between conditions ( t test ; n = 15 ) . ten - point scale hunger ratings did not differ between conditions , showing the expected decline from baseline ( detemir , 4.87 0.68 ; human insulin , 5.93 0.54 ; p > 0.11 ) and post - eeg recording values ( 5.87 0.62 vs. 5.93 0.56 , p > 0.91 ) to low postingestion levels ( 1.47 0.43 vs. 1.00 0.24 , p > 0.33 ; p > 0.12 for treatment time ) . a comparable pattern without significant differences between conditions was observed for thirst ( all p > 0.12 ) and tiredness ( p > 0.33 ) ratings . according to the bipolar questionnaire , subjects in the detemir compared with the human insulin condition felt more critical ( critical - comfortable , 2.87 0.13 vs. 3.27 0.18 , p < 0.009 ) and reported increased wakefulness ( sleepy - awake , 3.33 0.23 vs. 2.73 0.23 , p < 0.03 ) and hunger ( full - hungry , 4.20 0.20 vs. 3.53 0.22 , p < 0.01 ) immediately after infusions . the remaining dimensions and the post food intake assessment were not affected . the mood adjective checklist did not yield significant differences between conditions for any of the subscales . administration of insulin to the central nervous system reduces food intake ( 3,5 ) and body weight ( 4,6 ) . we demonstrate that while eliciting comparable peripheral effects , euglycemic infusion of insulin detemir compared with human insulin triggers a distinct negative shift in eeg dc - potential recordings and reduces calorie uptake in healthy men , supporting our hypothesis that detemir affects brain functions to a greater extent than human insulin and induces stronger anorexigenic effects on central nervous networks that control food intake . this outcome suggests that enhanced catabolic insulin signaling to the brain may be an important mechanism behind the limitation of weight gain observed in diabetic patients receiving detemir treatment ( 9,1114 ) . in accordance with other investigators ( 15,17 ) , we administered insulin doses that were considerably higher in the detemir than in the human insulin condition to compensate for the delayed onset that human insulin accordingly , timing and strength of the effects of detemir and human insulin on systemic glucose homeostasis as reflected by the rates of glucose infusion as well as blood glucose and serum glucagon concentrations were identical . c - peptide concentrations likewise did not differ between conditions during insulin infusion , merely showing a slight increase in the human insulin condition after the test buffet that may have been due to greater food intake in this compared with the detemir condition . congruent peripheral effects were also indicated by comparable serum leptin concentrations that are known to respond to insulin infusion ( 27,28 ) . on the background of equipotent systemic effects , detemir elicited a marked brain response as indicated by a widespread negative shift of scalp - recorded dc potentials that started around 15 min after detemir bolus injection and exceeded potential levels in the control condition that remained unaffected by human insulin infusion . in the human insulin condition , subjects received roughly the same total amount of insulin that , when administered in single bolus form , induced a negative dc - potential shift in foregoing experiments ( 21 ) . slowly infused over the course of 90 min , this dose obviously was too weak a stimulus to evoke dc - potential responses to human insulin in the present experiments . in contrast , the 90-min infusion of a detemir dose equivalent in terms of systemic action triggered a sustained negative dc - potential shift comparable with the previously reported effect of human insulin administered in high - dose bolus form ( 21 ) . this pattern indicates that although central nervous detemir effects may be mimicked by disproportionally high doses of human insulin , the relative impact of detemir on brain functions is considerably greater when both insulins are administered at doses with similar peripheral impact . the mechanisms behind the strong effect of detemir on scalp - recorded dc potentials can not be derived from our data . brain dc - potential shifts of this amplitude most likely reflect changes in extracellular ionic concentrations stemming from potential shifts at glial membranes that are endowed with receptors for insulin and igf ( 21,2931 ) . the assumption of a widespread effect on cerebral cellular networks also fits with the global nature and long duration of the dc - potential shifts induced by detemir infusion . our observations corroborate previous findings of increased brain responses to detemir in comparison with human insulin in animals ( 32 ) and humans ( 1517 ) . superior central nervous efficacy of detemir may be due to improved permeation of the lipophilic molecule into the brain compartment , with enhanced receptor - mediated blood - brain barrier transport of albumin - bound detemir adding to this effect ( 15,32 ) . the detemir - induced negative dc - potential shift that is presumably of primary glial origin thus may be reinforced by electrical potentials generated in the course of receptor - mediated detemir transport across the blood - brain barrier ( 33 ) . a most remarkable finding of our study is the reduction of ad libitum food intake by around 300 kcal in the detemir compared with the human insulin condition in the presence of identical peripheral actions of both insulins . this pattern renders the contribution of systemic mediators to this effect highly unlikely , rather suggesting that enhanced central nervous insulin signaling in the detemir condition resulted in decreased caloric intake . the concept of insulin providing catabolic feedback on the body 's energy resources to brain networks that control energy homeostasis has been well established in animals ( 2,3 ) as well as in humans ( 5,6 ) . thus , the decrease in food intake after detemir compared with human insulin infusion suggests that the enhanced effect on brain functions indicated by the negative dc - potential shift particularly impacts the central nervous control of food intake . although because of methodological constraints , dc - eeg could not be recorded during food intake proper ( 34 ) , this interpretation is supported by the strong correlation between the reduction in calorie intake and the antecedent dc - potential effect elicited by detemir . it is also in line with animal experiments in which intravenous detemir compared with human insulin injections were associated with enhanced insulin receptor phosphorylation in hypothalamic and cerebrocortical tissue in conjunction with increased eeg - assessed cortical activity , whereas the activation of the insulin receptor signaling cascade was similar in muscle tissue and liver ( 32 ) . interestingly , detemir compared with human insulin infusion increased rather than decreased self - rated hunger before test buffet presentation , which may have been due to a biasing influence of enhanced wakefulness and activation after detemir infusion ( 35 ) . alternatively , this finding may also indicate that central nervous insulin exerts its anorexigenic effects via meal - related signals that contribute to the termination of a meal , but not by affecting hunger motivation per se ( 5 ) . weight gain is a frequent side effect when blood glucose levels of diabetic patients are normalized by insulin ( 36,37 ) , but is less pronounced in patients undergoing detemir therapy ( 9,1114 ) . the assumption that the use of detemir limits weight gain because its favorable safety profile decreases defensive snacking to prevent hypoglycemia was not supported by comparative clinical studies showing that insulin glargine , which like detemir reduces the risk of hypoglycemia , is associated with greater weight gain than detemir ( 38,39 ) . against this background , the anorexigenic brain impact of detemir found in our study rather suggests the contribution of central nervous mechanisms to the weight - sparing effect of detemir in the clinical context .

objective . the role of health professionals in the decision making process of patients is usually heard or seen from the perspective of the patients . this paper gives the usually silent and invisible health professionals voice and visibility . it describes their views and attitudes towards reproduction by couples who are hiv positive and attempts to understand their perspectives . methods . in - depth interviews were conducted with twelve health professionals at an opportunistic infections clinic . transcribed interviews were analysed using the grounded approach to identify patterns and themes concerning views and attitudes of health professionals towards reproduction by hiv positive people . results . the study found that most health professionals generally had a negative attitude towards childbearing by hiv positive couples . their views and approaches on the issue were based mainly on biomedical considerations . the main discourses on childbearing that emerged from the study were the conditional choice , the antichildbearing , and the prorights . conclusion . most of the health professionals interviewed tend to take a generally negative stance towards reproduction by people with hiv / aids . there is a need for a clear set of guidelines for health professionals ( hps ) on how to deal with hiv positive people who may desire to reproduce . objectivein the treatment of diabetic patients , the long - acting insulin analog insulin detemir is less prone to induce weight gain than other insulin formulations . assuming that because of its pharmacologic properties , detemir displays stronger central nervous anorexigenic efficacy than human insulin , we compared acute effects of human insulin and detemir on electroencephalography ( eeg ) measures and food intake.research design and methodsfrontocortical eeg direct current ( dc ) potentials were recorded in 15 healthy men during two hyperinsulinemic - euglycemic clamps that included an insulin bolus injection ( human insulin , 17.75 mu / kg body wt ; detemir , 90 mu / kg body wt ) followed by a steady 90-min infusion ( 1.0 vs. 2.0 mu kg1 min1 ) . a higher dosage was chosen for detemir to compensate for its delay in impact relative to human insulin and to elicit similar systemic effects . at 20 min after infusion , subjects were allowed to eat ad libitum from a test buffet.resultsmean glucose infusions to maintain euglycemia ( p > 0.93 ) and blood glucose concentrations ( p > 0.34 ) did not differ between conditions . detemir infusion induced a negative dc - potential shift , averaging 372.2 v from 21 to 90 min that was not observed during human insulin infusion ( 146.5 v , p = 0.02 ) . detemir , in comparison with human insulin , reduced subsequent food intake by 303 kcal ( 1,257 vs. 1,560 , p < 0.04).conclusionswhile inducing comparable peripheral effects , detemir exerts stronger acute effects on brain functions than human insulin and triggers a relative decrease in food consumption , suggesting an enhanced anorexigenic impact of detemir compared with human insulin on central nervous networks that control nutrient uptake .

to increase blood safety , we introduced minipool nat screening in our blood donor service in 1997 for hepatitis b virus ( hbv ) , hepatitis c virus ( hcv ) , and hiv-1 and in 2000 for hepatitis a virus ( hav ) and parvovirus b19 ( 4 ) . for these purposes , 100-l aliquots of up to 96 blood samples were pooled . the complete pool of up to 9.6 ml was centrifuged at 58,000 g for 60 min at 4c . viruses were extracted by using spin columns , and nucleic acid was eluted in a total volume of 75 l . only 60 l of extract is needed for routine nat screening . a residual volume of 15 l can then be used for additional nat testing ( 5 ) for influenza viruses . the real - time quantitative amplification of influenza / h5 was performed according to the manufacturer s instructions ( artus influenza / h5 lc rt - pcr kit , qiagen , hamburg , germany ) by using a thermocycler ( lightcycler ; roche applied science , mannheim , germany ) . the specificity of this reaction was demonstrated for all subtypes of influenza a ( h1h15 , n1n9 ) and all subtypes of influenza b. samples with a positive test result in the first pcr were analyzed in a second pcr with influenza ( h5n1)specific primers and probes . therefore , the assay allows differentiation between avian influenza ( h5n1 ) and other influenza virus strains . to mimic a situation like an h5-positive donation , a purified culture supernatant of vero cells infected with influenza ( h5n1 ) ( strain a / thailand/1 ( kan-1)/2004 ) ( 6 ) was used as an external quantification standard . the viral rna concentration was determined in an external laboratory by multiple quantitative real - time pcr determinations ( 7 ) . different dilutions of the external influenza ( h5n1 ) subtype quantification standard ( 0.0 , 0.91 , 1.96 , 3.91 , 7.81 , 15.63 , 31.25 , 62.5 , 125 , and 250 pfu / ml ) were prepared , and 100 l of each dilution was spiked into 9.5-ml negative plasma pools . each dilution was repeatedly spiked and tested in 8 minipools . five microliters of the extract was analyzed with the generic influenza nat as well as with the specific influenza ( h5n1 ) nat . probit analysis of these data yielded a detection probability of > 95% in parallel tests when an average of at least 13.4 pfu / ml ( 95% confidence interval [ ci ] 8.3184 pfu / ml ) and 7.4 pfu / ml ( 95% ci 5.214.7 pfu / ml ) for influenza generic assay and for the influenza ( h5n1)specific test , respectively , were present in individual plasma samples before pooling . * influenza ( h5n1 ) standard was extracted from 9.6 ml of 96 pooled donor samples after centrifugation . the 95% detection limit was 13.4 pfu / ml ; the 50% detection limit was 4.8 pfu / ml . * influenza ( h5n1 ) standard was extracted from 9.6 ml of 96 pooled donor samples after centrifugation . the 95% detection limit was 7.4 pfu / ml ; the 50% detection limit was 2.5 pfu / ml . a total of 117 routine minipools , representing 10,272 blood donor samples , containing an average of 88 8 samples per pool , had previously been tested for hiv-1 , hbv , hcv , hav , and parvovirus b19 . all pools were negative for influenza virus when tested with the generic influenza pcr and the influenza ( h5n1)specific pcr . one pool had invalid results ( failed amplification of internal control rna , representing 0.01% of all analyzed runs ) . ( 8) , nat significantly increased the safety of blood products . at the german red cross , look - back examinations showed only 1 transfusion had transmitted hiv-1 ( 1998 ) after the introduction of nat testing . blood donor screening by nat was made technically and financially feasible by creating minipools of up to 96 individual samples per pool . roth et al . demonstrated an efficient enrichment for all tested viruses in plasma samples ( 9 ) . in the absence of an infective donor , different concentrations of the new influenza genotype h5n1 were spiked into minipools of 95 samples . as shown in table 2 , the influenza ( h5n1 ) subtype was detected by the generic influenza primers as well as by the influenza ( h5n1)specific primers when our routine minipool screening procedure was used . sensitivity was expressed as pfu / ml and can be converted into viral genome copy number according the calculation of yoshikawa et al . therefore , the analytical sensitivity was 804 geq / ml and 444 geq / ml for a generic influenza and for the influenza ( h5n1 ) subtype , respectively . after screening 10,272 samples by minipool nat , none of the samples were found to be infected by influenza , which corresponds with the low eiss index ( european influenza surveillance scheme index ) of < 20 during the study period ( february april , 2006 ) ( 10 ) . an eiss index > 80 is expected during an influenza epidemic , as was seen in 2005 . as with other viruses , a viremic phase of infection can be assumed to precede clinical symptoms such as fever ( 13,14 ) . ( 15 ) detected influenza ( h5n1 ) virus ( 3,080 copies / ml ) in the plasma of a 5-year - old boy , which indicates a viremic phase of influenza ( h5n1 ) infection . in addition to quarantine of infected patients , treatment with antiviral drugs , and development of avian influenza vaccines , blood donors should be tested during a pandemic to avoid transfusion - transmitted infections . our study demonstrates that nat screening could be incorporated into blood testing without delay and that the influenza virus could be sufficiently enriched by centrifugation . sensitivity of our influenza - screening method would have been sufficient to detect recently reported virus concentrations in plasma of infected persons ( 15 ) . however , as with all minipool methods , infections can be transmitted to transfusion recipients on rare occasions because the viremia level in the donor is below the analytical sensitivity of the screening assay . to reduce this risk , a selective infectious dose nat strategy ( e.g. , triggering of infectious dose nat testing when at least 1 viremic donation is collected per week with the standard minipool screening algorithm ) , as performed for west nile virus ( wnv ) screening in the united states implementation of wnv - nat in the united states in 2003 interdicted well over 1,000 donations from persons infected with wnv and is a good example of successful implementation of nat screening for emerging viruses . the collective fight against new viruses such as severe acute respiratory syndrome virus , wnv , or influenza ( h5n1 ) presents an immense challenge for the whole community , but new molecular - biologic methods offer opportunities to overcome this challenge . nat screening tests are now available soon after the sequencing of new viruses . in the absence of a general pathogen inactivation method for all blood products ( erythrocytes , platelets , and plasma ) small cell lung cancer ( sclc ) accounts for 1320% of all lung cancer diagnoses and is commonly classified into two stages , limited and extensive , according to the veteran 's administration lung cancer study group ( valg ) classification system . this system is used because most sclc patients present at a stage for which surgery is not appropriate , and thus are usually unable to be classified by the more commonly used cancer staging classification system , tumor - node - metastasis ( tnm ) , which requires surgical confirmation to achieve an accurate classification . sclc is characterized generally by a rapid growth rate , initial sensitivity to chemotherapy and radiation , and early metastasis to regional lymph nodes and/or distant sites . limited sclc is generally described as disease limited to one hemithorax , while extensive sclc is described as disease present in both hemithoraxes and/or metastasized to more distant areas of the body . those with limited stage sclc have a better prognosis than those with extensive stage disease . the median survival for limited sclc patients is 23 months , while those with extensive sclc have a median survival of 812 months , if treatment is administered . over 50% of lung cancer patients in canada are diagnosed at 70 years of age or older while over 20% are diagnosed at age 80 years or older . the standard of care for patients with sclc combined concurrent chemoradiotherapy for those with limited disease and chemotherapy alone for those with extensive disease . the preferred chemotherapy regimen is etoposide plus cisplatin ; however , etoposide plus carboplatin is an acceptable alternative for patients who are unable to tolerate cisplatin . many elderly sclc patients are not selected to receive chemotherapy , however , for fear of toxicity due to their age and the presence of comorbidities [ 7 , 8 ] . it is also recommended that patients who have achieved remission or stable disease after the completion of primary treatment receive prophylactic cranial radiation ( pci ) to reduce the risk of brain metastases . additionally , if a patient is not a good candidate for or refuses chemotherapy , they may receive radiation to symptomatic sites . evidence - based care for the elderly lung cancer population is lacking due to the underrepresentation of this population in clinical trials . cancer trials tend to consider 70 years of age as the reference point for being elderly ; however , there is not a specific minimum age that clearly defines the term . the tendency to exclude elderly patients from cancer treatment clinical trials on the basis of chronological age is largely because older patients are more likely to have serious comorbid conditions and have reduced organ function which can lead to higher drug - related toxicities [ 1114 ] . many elderly people , however , do not have any measurable loss of functional capacity and are free from significant medical problems . in canada , it has been estimated that 45% of those 7584 years of age and 22% of those 85 years of age and older are in overall good health . in addition , studies have shown that age alone is not significantly associated with adverse prognosis [ 7 , 17 ] . this suggests that elderly patients should not be excluded from therapeutic opportunities solely on the basis of age [ 18 , 19 ] . the care of elderly patients is , however , often complicated by comorbidities , frailty , and decreased organ function . the purpose of this study was to describe the receipt of chemotherapy provided to elderly patients with sclc in alberta , canada , and assess their chemotherapy tolerance and survival . we also sought to identify the reasons for not recommending chemotherapy and for dose reductions and assess the relationship of patient age in these decisions . in the absence of clear evidence from clinical trials , the analysis of the elderly sclc population through retrospective population - based studies such as this one helps assess and quantify the value of treating elderly cancer patients with chemotherapy . a retrospective , population - based study was conducted on all residents of alberta , canada , diagnosed with sclc at the age of 75 years or older in years 20042008 who had an oncologist - consult . selection of 75 years was chosen because the median patient age for sclc in alberta is about 70 years , and we wanted to focus on the significant minority of elderly patients . furthermore , on basis of our clinical experience , we felt that patients who are 75 years and older are the ones for whom ideal treatment is the least clear ; we , therefore , selected 75 years as the age cut - off . the province of alberta consists of an area of 660,000 km and has a population of 3.7 million . the healthcare system in alberta is funded and administered publically , as it is throughout all of canada ; standard cancer care such as consultations with specialists and chemotherapy is free to residents . consultations with oncologists , nonsurgical cancer treatment , and other services are provided at cancer care facilities . prior to receiving chemotherapy , a patient must be referred to one of six cancer facilities in the province to have a consultation with an oncologist with whom treatment options are discussed . two of these cancer facilities are located in the major cities of edmonton and calgary ; the remaining four are located in smaller cities . the alberta cancer registry was used to identify patients 75 years of age or older diagnosed with sclc ( international classification of diseases for oncology ( icd - o-3 ) topography codes c34.0c34.9 and morphology codes 80408045 ) in years 20042008 . patients were excluded if they were not residents of alberta , were diagnosed with combined small cell and nonsmall cell lung cancers , or had another cancer diagnosis for which they were receiving treatment . gender , date of birth , and date of death were also obtained from this source . the alberta cancer registry has been repeatedly recognized by the north american association of central cancer registries ( naaccr ) for its high level of completeness and for the timeliness of its data reporting . a chart review was conducted on all potentially eligible patients identified from the cancer registry to identify those who had an oncologist - consult and to obtain details of the chemotherapy received . the following data were extracted from patient charts : cancer stage ; presence and type of comorbidities ; eastern cooperative oncology group ( ecog ) performance status ; whether or not chemotherapy was recommended by the oncologist ; reasons for not recommending chemotherapy ; whether chemotherapy was administered or patient refused reasons patient refused chemotherapy ; chemotherapy start date ; treatment regimen received ; whether the patient received all chemotherapy cycles ; number of cycles received if less than the complete amount ; reasons for incomplete chemotherapy cycles ; whether dose reduction occurred ; reasons for dose reduction and changes to the initially recommended chemotherapy regimen . a complete course of chemotherapy was defined as receiving any of the regimens once every three weeks for 4 cycles . dose reduction was defined as any reduction in the dose of chemotherapy administered to the patient , compared to the recommended dose ; dose reduction that occurred at any time during chemotherapy treatment , including the first cycle , was included . information about other treatments received , such as radiation or second - course treatment , were not collected ; however , it is likely that most , if not all , patients with limited stage disease who received chemotherapy also received radiation . patients with extensive stage disease , however , would only have received radiation to relieve symptoms ; such treatment is palliative and would not impact survival . descriptive statistics were calculated to describe the utilization and tolerance of chemotherapy in the sclc patients who had an oncologist - consult . exploratory data analyses were performed to determine cut - off values for continuous variables and to assess the relationships of these variables with commencing chemotherapy , dose reductions , and not completing all chemotherapy cycles . chi - square test or fisher 's exact test ( if the expected value of a cell was less than 5 ) were performed to assess the statistical significance of these associations . kaplan - meier ( k - m ) curves were generated to compare patient survival by age ( < 80 versus 80 ) and treatment completeness status . in order to ensure chemotherapy , completeness status was known at the beginning of the time period ; the start time ( t0 ) was defined as 12 weeks after the oncologist - consult ; all patients were followed to the earlier of their death date or december 31 , 2010 . the log - rank test and the wilcoxon test were used to determine statistical differences between the curves . cox proportional hazard models were used to estimate the effect of treatment status on patient survival , adjusting for ecog score , disease stage , age at diagnosis , number of comorbidities , and chemotherapy regimen used . as in the k - m graphs , the start time ( t0 ) was defined as 12 weeks after the oncologist - consult date in order to categorize patients properly for chemotherapy completion and dose reduction statuses . the wald chi - square test was used to calculate p values for the hazard ratio estimates . sas software ( version 9.2 ; sas institute , cary , nc , usa ) was used to perform all analyses . there were 238 patients aged 75 years or older diagnosed with sclc in alberta , canada , in years 2004 to 2008 . of these , 11 were excluded for the following reasons : 2 were not residents of alberta ; 7 had a diagnosis of combined non - small cell and small cell lung cancer ; and 2 had another cancer diagnosis for which they were receiving treatment . of the remaining 227 potentially eligible patients , 171 ( 75% ) had an oncologist - consult to discuss treatment options and were included in this study . there were 56 patients ( 25% of those potentially eligible for this study ) who did not have an oncologist - consult . relative to the patients who had an oncologist - consult , those who did not tend to be older ( 46% were older than 80 years compared to 35% ) . additionally , almost half of them ( 46% ) died within two weeks of their diagnosis . figure 1 shows a flow chart of the proportion of patients for whom chemotherapy was recommended , began chemotherapy , refused it , received regimen , completed chemotherapy , and who received versus who did not receive chemotherapy cycles at the full dose . of the patients who had an oncologist - consult , 84% were recommended chemotherapy and 68% began chemotherapy ( including one patient who commenced chemotherapy despite the fact it is not recommended ) . the chemotherapy regimens received were cisplatin / etoposide , carboplatin / etoposide , cyclophosphamide / adriamycin / vincristine ( cav ) , and oral etoposide . the regimens most frequently used were combinations carboplatin / etoposide ( 47% ) and cisplatin / etoposide ( 31% ) . of those who began chemotherapy , 52% completed all cycles , 66% did not have any dose reductions , and 31% completed all cycles at the recommended dose . of those who completed all cycles of chemotherapy , 34% had limited stage disease . the relationship between the demographic and clinical characteristics of the patients included in the study and age is shown in table 1 . just over half of the patients in both age groups were male , and 77% had extensive disease . a higher percentage of patients 80 years and older had an ecog score of 3 or 4 compared to those 7579 years , 47% versus 39% , respectively , p = 0.18 . patients 80 years and older were more likely to have two or more comorbidities than those aged 7579 , 62% versus 48% , respectively . table 2 displays the demographic and clinical characteristics of the patients included in the study and the relationship between those characteristics and beginning chemotherapy , having a dose reduction , and incomplete chemotherapy cycles . of the patients included in the study , 57% were male , 35% were 80 years or older , 77% had extensive disease , 42% had an ecog score of 3 or 4 , and 24% had 3 or more comorbidities . a higher percentage of patients aged 7579 received chemotherapy than those 80 years or older , 74% versus 58% , respectively , p = 0.15 . of those who received chemotherapy , a higher percentage of patients 80 years or older had dose reductions than those 7579 years , 46% versus 29% , respectively , p = 0.09 . just over half of patients in both age groups , however , received all cycles of chemotherapy . patients with limited disease were more likely to receive chemotherapy than those with extensive disease ( 87% versus 63% , p = 0.02 ) ; of those who received chemotherapy , 52% ( 43 patients ) with extensive disease did not complete all chemotherapy cycles compared to 36% ( 12 patients ) with limited stage disease , p = 0.13 . similarly , patients with a poor performance status ( ecog 3 or 4 ) were less likely than those with a good performance status to complete all cycles of chemotherapy , 65% versus 37% , p = 0.007 . of those who had an oncologist - consult , 28 patients ( 16% ) were not recommended chemotherapy , and 27 patients ( 19% ) for whom chemotherapy was recommended refused it . oncologists ' reasons for not recommending chemotherapy and patients ' reasons for refusing it are listed in table 3 . the most common reason oncologists indicated for not recommending chemotherapy was patient performance status ( 22 of 28 patients ) . the second most common reason was the presence of comorbidities ( 16 of 28 patients ) . most patients who refused chemotherapy did so due to concerns about toxicity ( 20 of 27 patients ) . of those who began chemotherapy , the most common reason for dose reduction was hematological toxicity ( 30 of 40 patients ) , while 10 of 40 patients had a dose reduction due to frailty and performance status ( table 4 ) . similarly , receipt of an incomplete number of chemotherapy cycles was largely attributable to hematological toxicity ( 32 of 56 patients ) , concerns regarding frailty and performance status ( 25 of 56 patients ) , and non - hematological toxicity ( 19 of 56 patients ) . table 5 outlines the drug regimens received and the number of cycles completed by treatment status . patients who received the carboplatin / etoposide regimen were twice as likely to have dose reductions as those who received oral etoposide , 44% versus 20% , respectively , p = 0.13 . conversely , those who received the oral etoposide regimen were more likely to receive an incomplete number of chemotherapy cycles than those who received the combined etoposide / platin - based regimens , 64% versus 43% , respectively , p = 0.05 . of those who began chemotherapy , 61 patients ( 52% ) completed all cycles ; 25 ( 41% ) of those who completed all cycles had a dose reduction . a large proportion ( 26% ) of people who began chemotherapy only completed one cycle even though 20% of these people received it at a reduced dose . all but four patients died by the end of the follow - up period , december 31 , 2010 . lung cancer was the recorded cause of death for all patients with the exception of 14 ; 13 patients died of a noncancer related cause , and one patient died of prostate cancer . figure 2 displays k - m survival curves by chemotherapy cycle completion status ( complete versus incomplete / did not receive ) and age group ( 7579 versus 80 or older ) . those who completed chemotherapy had a better survival rate than those who did not ( p < 0.0001 ) . the median survival for those who did not complete chemotherapy was 3 months and 23 days , compared to 7 months and 13 days for those who completed all cycles . the survival rate of those who completed chemotherapy did not differ by age group ( p = 0.21 ) . table 6 presents the results from the adjusted survival analysis , generated from the cox proportional hazards model . the risk of death for patients who did not complete all cycles of chemotherapy was 2.72 ( 95% confidence interval : 1.52 to 4.87 ) relative to those who completed chemotherapy at full dose after adjusting for other variables ( p = 0.0007 ) . the risk of death for patients who completed treatment at a reduced dose did not differ from those who completed chemotherapy at the full dose ( hr = 1.02 , 95% confidence interval : 0.57 to 1.82 , p = 0.94 ) . due to the overlapping confidence intervals between the completed / reduced dose group and the not completed group , the model was rerun using the completed / reduced dose group as the reference . in this analysis , the risk of death for patients who did not complete chemotherapy was 2.67 ( 95% confidence interval : 1.45 to 4.91 , p = 0.0016 ) similar to the results shown in table 6 using the completed / full dose group as the reference . the purpose of this study was to describe the uptake and tolerance of chemotherapy among elderly patients with sclc and assess their survival . these elderly patients , compared to those 7579 years of age , received less chemotherapy and were more likely to receive a dose reduction , but were equally likely to complete all chemotherapy cycles . notably , the adjusted hazard ratio of death did not differ between the two age groups . overall , 52% of patients who began chemotherapy completed all cycles , and 41% had reduced chemotherapy doses . these results are confirmation that a significant proportion of elderly patients are able to tolerate chemotherapy and receive a survival benefit from it even in the presence of dose reductions . our results also suggest that elderly patients who have their chemotherapy dose reduced but complete all chemotherapy cycles have a similar survival ( hr 1.02 , ci 0.571.82 ) to those who complete all chemotherapy cycles at the full dose , after adjusting for ecog score , disease stage , age , co - morbidity count , and drug regimen . several phase ii clinical trials have tested the efficacy of lower dose combinations of concurrent carboplatin and etoposide regimens in elderly sclc patients [ 2326 ] . despite some differences in treatment schedule and dosing , three of these studies reported similar survival results as the usual treatment regimen , with a median survival of 4146 weeks . one of these studies reported lower median survival , however , with a median survival of 37 weeks . further investigation is , therefore , warranted to better assess the relationship between chemotherapy dose reduction and survival of elderly sclc patients . a larger study similar to ours which included a population - based group of elderly sclc patients was conducted in the netherlands . the most common reasons for not receiving chemotherapy were a combination of age , co - morbidity , poor performance status , and refusal by the patient or family , which are similar reasons as those identified in our study . they found that 53% of sclc patients received chemotherapy , which is also similar to the 52% of potentially eligible patients from our study that received chemotherapy . as well , a similar percentage of patients had a dose reduction ( 30% versus 34% ) and were unable to complete all chemotherapy cycles ( 43% versus 47% ) in the netherlands study compared to the current study . although a large proportion of elderly patients were able to tolerate and experience a survival benefit from chemotherapy , 48% of patients who began chemotherapy were not able to complete all treatment cycles and did not have a survival benefit . the difficulty is identifying the ones who are ; there is a need for a reliable means to identify elderly patients who would benefit from chemotherapy that does not base its conclusions on chronological age and rather aims to determine biological age by the measurement of objective standard measures [ 6 , 19 , 28 ] . the use of a multidimensional geriatric assessment tool for this purpose has been suggested as a means of achieving this end [ 19 , 29 ] . suggested domains of the assessment tool include co - morbidity , functional status , emotional conditions and mental status , social support , polypharmacy , and nutritional state . even though the tool was first introduced over 15 years ago , the best form of the tool has yet to be defined [ 6 , 29 ] . further efforts are needed to optimize such a tool and implement it in routine practice . a limitation to the study is the nature of all retrospective studies in that they can not prove causality . additionally , there is always selection bias in terms of which patients receive treatment in a real clinical situation as opposed to a clinical trial setting . on the other hand , an inherent strength of a population - level retrospective study such as this one is that the treatment and outcome can be described for every patient , as we have done so herein . the major limitations are in obtaining complete information on all the factors that might impact one of or both treatment and outcome in order to properly adjust for them in analyses . in our study we had limited information on the patients who did not have an oncologist - consult , representing 25% of the entire population of sclc patients aged 75 years or older in alberta . the data we do have , however , suggests that many of these patients would not have been candidates for any kind of treatment as they died very soon after being diagnosed . it is possible , however , that some of them could have benefited from chemotherapy but did not have the opportunity because they were not referred to an oncologist , were unable to obtain transportation to an oncologist , were not interested in receiving chemotherapy , or another reason . we were not able to identify reasons for not seeing an oncologist . regarding the patients who did have an oncologist - consult , performance status was missing for 18% of patients , and we did not collect information on receipt of other treatment modalities which could have affected survival and the ability to tolerate chemotherapy . it is possible that there is also missing / incomplete information related to the specific reasons for dose reductions and incomplete cycles . a further limitation arises from the relatively small number of patients with limited disease , which prevented us from fully exploring the interrelationships between age , stage , chemotherapy uptake , tolerance , and survival . we have shown that while an appreciable proportion of elderly patients diagnosed with sclc do not begin chemotherapy treatment , those that do are able to tolerate the treatment and receive survival benefits from it . it is , therefore , vital that elderly patients as well as younger patients are considered for established treatment . our results also suggest that elderly sclc patients who complete chemotherapy at a reduced dose have a similar prognosis to those who receive the full dose . future research should focus on better understanding the relationship between frailty and toxicity to ensure the careful selection of patients who will benefit from chemotherapy treatment .

influenza viruses , including highly pathogenic avian influenza virus ( h5n1 ) , could threaten blood safety . we analyzed 10,272 blood donor samples with a minipool nucleic acid amplication technique . analytical sensitivity of the method was 804 geq / ml and 444 geq / ml for generic influenza primers and influenza ( h5n1 ) subtype specific primers . this study demonstrates that such screening for influenza viruses is feasible . the treatment of elderly cancer patients is complicated by many factors . we sought to assess the uptake and tolerance of chemotherapy among patients 75 years and older diagnosed with small cell lung cancer ( sclc ) in years 20042008 in alberta , canada , and assess their survival . all patients who met the above criteria and had an oncologist - consult were included . data were obtained from the alberta cancer registry and chart review . a total of 171 patients were included in the study , 117 ( 68% ) of whom began chemotherapy . of those , 52% completed all cycles , 66% did not have any dose reductions , and 31% completed all cycles at the recommended dose . the risk of death for patients who did not complete all cycles of chemotherapy was 2.72 ( 95% ci : 1.524.87 ) and for those who completed all cycles but with a reduced dose was 1.02 ( 95% ci : 0.571.82 ) relative to those who completed chemotherapy at full dose after adjusting for several demographic / clinical factors . our results suggest that a significant proportion of elderly patients are able to tolerate chemotherapy and receive a survival benefit from it while those who experience toxicity may receive a survival benefit from a reduction in chemotherapy dose as opposed to stopping treatment .

the major cause for the failure of primary endodontic therapy is the persistence of intra- and extra - radicular microbial infections . in every failed case , whenever possible , nonsurgical endodontic retreatment should be preferred in comparison with other radical interventions such as apical surgery or extraction because the nonsurgical retreatment is less invasive and has good long - term survival rates . during nonsurgical endodontic retreatment therapy , for the retrieval of filling materials , several techniques such as hand files , rotary files , and ultrasonic instruments either alone or in combination with heat or solvents have been used . these removal techniques are dependent on the anatomy , size and regions of the root canal , aged , overfilled or overextended , and/or underfilled or underextended fillings . a combination of methods is frequently preferred to achieve a safe , efficient , and potentially complete removal of the filling materials from the root canal system . gutta - percha along with different endodontic sealers is the most commonly used filling material . retrieval of gutta - percha usually does not require a great effort while using the aforementioned techniques either alone or in combination . however , various evidence - based studies till date have reported that filling material residues , especially sealers present on canal walls and in their microscopic ramifications , may either remain inaccessible or resist dissolution . in such cases , organic solvents play a vital role in the thorough removal of endodontic sealers . this facilitates the efficient disinfection of the root canal system and thus the probability of long - term treatment success . although chloroform is considered as the most effective solvent in nonsurgical retreatment , it has been banned by the u.s . food and drug administration in 1976 due to its carcinogenic potential and toxicity to the tissues . therefore , various alternatives such as halothane , rectified turpentine , endosol r and e benzene , methyl chloroform , ether , xylene , orange oil , and eucalyptol have been suggested and used for softening of filling materials . xylene ( dimethylbenzene ) is an aromatic compound and has been reported to be a very efficient solvent for root canal obturating materials . essential oils such as orange oil , eucalyptus oil , and pine oil are able to dissolve most of the endodontic sealers . they have been reported to be safe , biocompatible , noncarcinogenic , and useful for this purpose . therefore , they could influence and consequently determine the clinical effectiveness of the commonly used organic solvents . apexit plus has been studied very well regarding their solubility in various solvents . however , there are as yet no studies that show the effectiveness of the aforementioned solvents on endomethasone n and adseal root canal sealers . therefore , a simple , reproducible , and cost - effective laboratory study was carried out to comparatively evaluate the effectiveness of eucalyptus oil , orange oil , and xylene in dissolving endomethasone n , apexit plus , and adseal . standardized stainless steel molds ( 8 mm diameter , 2 mm height ) were used to prepare two hundred and forty samples ( eighty for each endodontic sealer ) . endomethasone n ( septodont , saint maur , france ) , apexit plus ( ivoclar vivadent , schaan , liechtenstein ) , and adseal ( meta biomed co , cheongju , korea ) had been used in this study and their compositions are shown in table 1 . composition of different endodontic sealers as provided by manufacturers each sealer was mixed according to the manufacturer 's instructions . freshly mixed materials were carefully poured into the sample molds placed on a glass slab using a 2 ml syringe to prevent air entrapment , and a microscope slide covered with cellophane strip was then pressed onto the upper surface to make the surface flat . all samples along with their steel molds were then transferred to a chamber with 80% relative humidity and 37c temperature and left untouched to set sealers for 48 h and then removed from the chamber . the samples were weighed in grams ( up to four decimal places ) 3 times on an analytical balance ( sartorius te214s , sartorius ag , germany ) . the samples were then divided into four groups of twenty each for immersion in different organic solvents . solvents used in this study were eucalyptus oil , orange oil , xylene , and distilled water ( negative control group ) . each group was further subdivided into two equal subgroups ( n = 10 ) for 2 and 10 min of immersion . at room temperature , all sealer samples were immersed completely in 20 ml of solvent stored in glass vials . after the specified immersion period ( 2 and 10 min ) , the samples were removed from glass vial , rinsed with 100 ml of triple distilled water , and then blotted dry with absorbent paper . samples were allowed to dry for 24 h at 37 1c in an oven and then kept in dehumidifier / desiccators . thereafter , the samples were weighed 3 times and the mean was calculated . the amount of sealer dissolved was determined as the difference between the original weight of the sealer and its final weight . data were subjected to statistical analysis of percentage weight loss for each root canal sealer in different solvents at different time intervals and summarized as mean standard deviation . all groups were compared by two factor analysis of variance , and significance of mean difference within and between the groups was done by tukey 's post hoc test . the mean percentage of weight loss of all sealers and their relative solubility in different organic solvents at 2 and 10 min are shown in table 2 and figure 1 . mean percentage with standard deviation ( ) of weight loss for each endodontic sealer in different organic solvents at 2 and 10 min dissolution of different endodontic sealers in different organic solvents in all the solvent groups , all the three sealers showed more weight loss at 10 min than 2 min . the weight loss was highest for endomethasone n followed by apexit plus and adseal at both the periods . in the control group ( distilled water ) , minimum and insignificant ( p > 0.05 ) weight loss was observed for all sealers at both time periods . in essential oils , both apexit plus and endomethasone n dissolved more ( p < 0.001 ) at 10 min as compared to 2 min . however , adseal showed no significant ( p > 0.05 ) difference in its solubility between the periods . in xylene , all sealers showed significantly ( p < 0.05 or p < 0.001 ) more weight loss at 10 min compared to 2 min . for each time period , comparing the effect of eucalyptus oil on sealers , tukey test showed significantly ( p < 0.05 or p < 0.001 ) different and higher weight loss for both apexit plus and endomethasone n as compared to adseal at both 2 and 10 min . dissolution of apexit plus and endomethasone n did not differ ( p > 0.05 ) at 2 min . however , at 10 min , weight loss for endomethasone n was significantly ( p < 0.001 ) higher than apexit plus . similarly , in both orange oil and xylene , endomethasone n dissolved significantly ( p < 0.01 or p < 0.001 ) more than both apexit plus and adseal at both 2 and 10 min . apexit plus and adseal showed similar behavior in both orange oil and xylene at 2 min . however , at 10 min , apexit plus dissolved significantly ( p < 0.001 ) more than adseal in both solvents . apexit plus and endomethasone n showed significantly ( p < 0.001 ) different and higher weight loss at 10 min as compared to 2 min in all solvents except distilled water . adseal showed significantly ( p < 0.001 ) different and higher weight loss in xylene at 10 min as compared to 2 min . however , in other solvents , dissolution of adseal was not significantly different ( p > 0.05 ) between 2 and 10 min . apexit plus dissolved significantly ( p < 0.01 or p < 0.001 ) more in eucalyptus oil , orange oil , and xylene as compared to distilled water at both 2 and 10 min . however , its dissolution pattern did not differ ( p > 0.05 ) among eucalyptus oil , orange oil , and xylene at both the time periods . endomethasone n and adseal showed significantly ( p < 0.001 ) different and higher weight loss in eucalyptus oil , orange oil , and xylene as compared to distilled water at both 2 and 10 min . at 2 min , their dissolution profile did not differ ( p > 0.05 ) among eucalyptus oil , orange oil , and xylene . however , at 10 min , both endomethasone n and adseal showed significantly ( p < 0.01 or p < 0.001 ) higher weight loss in xylene as compared to eucalyptus oil and orange oil , in which their dissolution was not statistically significant ( p > 0.05 ) . in all the solvent groups , all the three sealers showed more weight loss at 10 min than 2 min . the weight loss was highest for endomethasone n followed by apexit plus and adseal at both the periods . in the control group ( distilled water ) , minimum and insignificant ( p > 0.05 ) weight loss was observed for all sealers at both time periods . in essential oils , both apexit plus and endomethasone n dissolved more ( p < 0.001 ) at 10 min as compared to 2 min . however , adseal showed no significant ( p > 0.05 ) difference in its solubility between the periods . in xylene , all sealers showed significantly ( p < 0.05 or p < 0.001 ) more weight loss at 10 min compared to 2 min . for each time period , comparing the effect of eucalyptus oil on sealers , tukey test showed significantly ( p < 0.05 or p < 0.001 ) different and higher weight loss for both apexit plus and endomethasone n as compared to adseal at both 2 and 10 min . dissolution of apexit plus and endomethasone n did not differ ( p > 0.05 ) at 2 min . however , at 10 min , weight loss for endomethasone n was significantly ( p < 0.001 ) higher than apexit plus . similarly , in both orange oil and xylene , endomethasone n dissolved significantly ( p < 0.01 or p < 0.001 ) more than both apexit plus and adseal at both 2 and 10 min . apexit plus and adseal showed similar behavior in both orange oil and xylene at 2 min . however , at 10 min , apexit plus dissolved significantly ( p < 0.001 ) more than adseal in both solvents . apexit plus and endomethasone n showed significantly ( p < 0.001 ) different and higher weight loss at 10 min as compared to 2 min in all solvents except distilled water . adseal showed significantly ( p < 0.001 ) different and higher weight loss in xylene at 10 min as compared to 2 min . however , in other solvents , dissolution of adseal was not significantly different ( p > 0.05 ) between 2 and 10 min . apexit plus dissolved significantly ( p < 0.01 or p < 0.001 ) more in eucalyptus oil , orange oil , and xylene as compared to distilled water at both 2 and 10 min . however , its dissolution pattern did not differ ( p > 0.05 ) among eucalyptus oil , orange oil , and xylene at both the time periods . endomethasone n and adseal showed significantly ( p < 0.001 ) different and higher weight loss in eucalyptus oil , orange oil , and xylene as compared to distilled water at both 2 and 10 min . at 2 min , their dissolution profile did not differ ( p > 0.05 ) among eucalyptus oil , orange oil , and xylene . however , at 10 min , both endomethasone n and adseal showed significantly ( p < 0.01 or p < 0.001 ) higher weight loss in xylene as compared to eucalyptus oil and orange oil , in which their dissolution was not statistically significant ( p > 0.05 ) . during nonsurgical endodontic retreatment , the removal of root canal obturating materials by purely mechanical means may lead to root perforation , canal straightening , or alteration of the original canal outline . therefore , the use of organic solvents has been anticipated to decrease the resistance of obturating materials inside the canal so that their retrieval can become easier in synergism with instrumentation . however , clinicians should be very cognizant about the use of removal techniques and materials for the dismantlement of the previous treatment because very strong solvents are capable of softening the enamel and dentin , which may promote canal transportation and chemical pericementitis if penetrated beyond the apex . there exist no specific standards for the measurement of root canal sealer solubility in organic solvents . various studies have reported that a mean time of 1.510.8 min is required for the removal of very well - compacted obturating materials by instrumentation with or without solvents . in the present study , application of 2 and 10 min for standardized samples allowed us to demonstrate clear differences in the solubility profiles of the endodontic sealers used in various solvents . to enhance the accuracy of the measurements , one sealer sample was used for just one immersion period , thus excluding its undesirable weight loss because of the repeated drying and immersion . after the specified immersion period , all samples were washed with distilled water to remove the decomposed loosen debris . although several studies regarding the solubility of endodontic sealers in various organic solvents have been reported in the literature , the comparison of the effects of sealers on solvents and vice versa was found interesting in this laboratory analysis . all endodontic sealers used in this study were found to be soluble to some extent in all the four solvents . xylene was the more effective solvent followed by the essential oils ( eucalyptus and orange oil ) and distilled water the least . moreover , the effect of all solvents was found gradual and proportional to the contact time with the surface of the sealers . organic solvents are usually employed in paint industries to soften the coated materials for their easy removal . these solvents can be applied in retreatment therapy for the retrieval of resistant or strongly adhered sealers in canal irregularities and ramifications . several organic solvents have been investigated in the literature for their softening or dissolution capability in endodontic retreatment . since all solvents are known for their toxicity to tissues to variable extent , the selection of an ideal organic solvent for any nonsurgical endodontic retreatment requires an equilibrium between its sealer dissolution competency and clinical safety regarding toxicity and belligerence toward the tissues . the use of essential oils in endodontics is growing because of their safety , biocompatibility , and noncarcinogenicity . eucalyptus oil is the distilled oil obtained from the leaves of eucalyptus globulus ; a plant of the family myrtaceae native to australia and cultivated worldwide . orange oil , an extract of the peel of sweet orange fruit ( citrus sinensis ) is an efficient alternative to potentially toxic solvents . sweet orange oil consists of approximately 90% d - limonene ; a solvent used in various household products . d - limonene is an efficient cleaning agent with a pleasant smell and is considered environment friendly . there is no evidence for its carcinogenicity or genotoxicity . further , orange oil is less cytotoxic and more biocompatible than eucalyptol and chloroform . owing to the concerns about the carcinogenicity of chloroform and unpredictable , idiosyncratic hepatic necrosis and toxicities of halothane , xylene and essential oils can be considered as safe alternatives . it is commonly considered as an efficient solvent for several organic materials such as gutta percha ( alkadienes ; a hydrocarbon ) , polymers ( resilons ; a polycaprolactone ) , resins and sealers . chloroform dissolves rather than softens the obturating materials , leaving residues on the canal walls and its fast evaporation makes use of more and more solvent , which makes its use messy and inconvenient . however , the action of xylene is relatively slow , which permits an efficient and biologically safe removal of softened filling materials . grduysus et al . reported that xylol showed similar behavior in dissolving gutta - percha as chloroform . in the present study , it has been reported that both xylol and orange oil were excellent in softening the gutta - percha and sealer cements without any deleterious effects . orange oil has been shown to be more biocompatible than eucalyptol , xylol , chloroform , and halothane . eugenol - based sealers was found similar to that of eucalyptol , chloroform , and xylol . however , orange oil and eucalyptol are poor to dissolve the resin - based materials . mushtaq et al . and martos et al . have stated that dissolving capacity of orange oil is inferior to chloroform and xylene . however , this is in contrast to some studies which showed similar dissolution behavior of orange oil , eucalyptol , chloroform , and xylol . stated that xylol was the best solvent whereas chloroform , eucalyptol , and orange oil presented a similar solvent profile . this indicates that both eucalyptol and orange oil could be suitable as solvents for softening or dissolving the endodontic sealers . the dissolution capacity of eucalyptol is slower than that of other commonly used solvents at room temperature . furthermore , this solvent can be easily washed out after retrieval of filling materials by irrigation with tensio - active solutions ( sodium laurel sulfate ) . under the experimental conditions of the present study , adseal ( an epoxy resin - based material ) it may be due to the fact that epoxy resin ( adseal ) is a heavily cross - linked , rigid , and strong polymer . adseal showed significantly higher dissolution in xylene at 10 min than eucalyptus and orange oils . interestingly , no significant dissolution was observed in eucalyptus oil and orange oil at both time periods . bodrumlu and kayaoglu in their in vitro study concluded that ah plus ( epoxy resin - based sealer ) dissolved to some extent and more than ketac - endo ( glass ionomer - based sealer ) , using either eucalyptus oil or chloroform as the solvent . martos et al . reported that the efficacy of eucalyptus oil , orange oil , xylene , and chloroform to dissolve ez - fill ( epoxy resin - based sealer ) was not significantly different from each other at 2 min . apexit plus , a calcium hydroxide - based sealer has a more convenient delivery form and hydrophilic formulation of apexit . consequently , the material is more reliable if used in thicker layers . in this investigation , apexit plus had shown more pronounced dissolution in all the organic solvents as compared with distilled water . no significant difference in its dissolution was observed among eucalyptus oil , orange oil , and xylene at both time periods . the reason for low solubility could be the more consistent setting reaction forming a more stable complex . apexit plus has a ph of < 8 , which is not true for other calcium hydroxide - based root canal sealers . this low ph may be because of less hydroxyl ion concentration which reacts with the acidic phenol group of salicylates subsequent to the dissociation of calcium hydroxide . mushtaq et al . reported that apexit plus showed the maximum weight loss in xylene . however , its dissolution in refined orange oil and tetrachloroethylene was found to the same extent . whitworth and boursin reported that apexit was significantly more soluble in halothane and chloroform as compared to ketac - endo and ah plus . there are no published data regarding the action of other organic solvents on this material . therefore , the effective retrieval of this material from the root canal system using these organic solvents can not be assured . it has been developed to ameliorate the neurotoxic and mutagenic effects of paraformaldehyde present in its predecessor . its cytotoxicity is approximately 30 times lower than a formaldehyde - containing sealer . in this study , it showed a higher weight loss than other endodontic sealers and the maximum in xylene followed by orange oil and eucalyptus oil . this high degree of solubility is probably due to the release of eugenol , and the hydrolysis of hardened zinc eugenolate . zoe - based materials decompose hydrolytically as follows : zn ( c10h11o2)2 + 2h2o = zn ( oh)2 + 2c10h11o2 this zinc hydroxide is almost insoluble in neutral , but highly soluble in an acidic solution . eugenol - based sealer ( endofill ) by orange oil , chloroform , and eucalyptus oil in a scanning electron microscope retreatment study . eugenol - based sealer presented significantly lower solubility in halothane than chloroform , which was 9 times more effective than eucalyptus oil when exposed for 20 min . the present laboratory investigation did not consider the clinically imposed parameters such as canal system anatomy , temperature , access , volume of exchange , dilution or displacement by biological fluids , or irrigants regarding the action of solvents on root canal sealing cement . therefore , its result can not be directly extrapolated to clinical scenarios . the search for more effective and biocompatible universal solvent should continue and should not be confined simply to the softening and gross removal of obturating materials . within the limitations of this laboratory study , the following conclusions had been drawn . xylene was the most effective solventorange oil and eucalyptus oil showed no significant difference in their solvent behaviorall the sealers showed more dissolution in all the solvents used at 10 min than 2 minin distilled water , insignificant sealer dissolution was observed . xylene was the most effective solvent orange oil and eucalyptus oil showed no significant difference in their solvent behavior all the sealers showed more dissolution in all the solvents used at 10 min than 2 min in distilled water , insignificant sealer dissolution was observed . trypanosoma cruzi is an obligate intracellular parasite that is responsible for chagas disease , which affects 1618 million people in latin america . this parasite has a complex biphasic life cycle in which four developmental forms alternate between the reduviid beetle vector ( epimastigotes and metacyclic trypomastigotes ) and the mammalian host ( amastigotes and bloodstream trypomastigotes ) . transmission is initiated in the reduviid beetle vector , which becomes infected by taking up circulating trypomastigotes during a blood meal . after trypomastigotes differentiate to epimastigotes in the insect gut lumen , the parasite divides by binary fission before migrating along the hindgut and rectum , where they transform to metacyclic trypomastigotes . these trypomastigotes are released near the bite wound with the insect feces during the next blood meal . following its introduction into mammalian blood , the trypomastigotes penetrate nonphagocytic and phagocytic cells through a parasitophorous vacuole to start the intracellular cycle . in this stage amastigotes develop into nondividing bloodstream trypomastigotes that can either initiate another round of infection to propagate to different organs or can be taken up by the insect vector to complete the life cycle . throughout its life cycle , t. cruzi survives under a wide range of environmental conditions that induce complex morphological changes among parasite stages . in addition to the four main developmental forms , it is possible to observe intermediate forms ( ifs ) that seem to follow the same differentiation path , independent of whether they exist in a vertebrate or in an invertebrate host [ 1 , 2 ] . intermediate forms appear transiently during the differentiation of epimastigotes into metacyclic trypomastigotes ( metacyclogenesis ) in the triatomine , the differentiation of metacyclic trypomastigotes ( primary amastigogenesis ) , and tissue - derived trypomastigotes ( secondary amastigogenesis ) into amastigotes and also into bloodstream trypomastigotes inside the mammalian host cell [ 25 ] . adaptation of t. cruzi to diverse environments found in the different hosts undoubtedly induces a complex regulation of gene expression that apparently precedes the morphological changes observed during parasite transformation . several researchers have studied some of the factors that represent physiological stress for the parasite and have demonstrated that temperature , nutritional conditions , and ph stimulate morphological differentiation during amastigogenesis [ 58 ] . the vast majority of the information regarding in vivo and in vitro amastigogenesis comes from data in which tissue - derived trypomastigotes were used because it was possible to obtain higher yields of these parasites [ 3 , 5 , 815 ] . ultra - structural and molecular analyses during the trypomastigote to amastigote transformation have shown a complex and progressive morphological rearrangement of parasite shape and flagellum that has been associated with the differential expression of stage - specific antigens [ 3 , 5 , 8 ] . a comparative morphology study showed that even when metacyclic and bloodstream trypomastigotes share similar biological and morphological properties , primary and secondary amastigogenesis apparently display different developmental processes , which suggests that their intracellular mechanisms are different . although the basic features of the amastigogenesis transformation process are known , the molecular mechanisms involved are still unidentified . analysis of the molecules implicated in the detonation and control of the transformation process will increase our knowledge about morphogenesis and gene expression programs that are involved not only in the differentiation between developmental forms but also during parasite transitions from the nonreplicative to the replicative stage . the usefulness of a differentiation system for molecular research in which a population of cells is involved depends first on the synchrony of the transition , second on the availability of easily analyzable markers for monitoring the process , and third on the system efficiency for obtaining large enough amounts of analyzable sample for further biochemical , biological , and molecular analyses . to date , in vitro conditions that allow for obtaining ifs that satisfy these needs have not been reported . therefore , in vitro conditions that induce high rates of pure ifs during the transformation of culture - derived trypomastigotes into amastigotes are described in this work . furthermore , morphological , cellular , and molecular characterizations of the different ifs obtained are presented . nih 3t3 fibroblasts were grown in high glucose dulbecco 's minimal essential medium ( hgdmem ) supplemented with 10% fetal bovine serum ( fbs ) , 1% glutamine , and 5 g / ml penicillin - streptomycin at 37c in a humidified atmosphere with 5% co2 in a 75 cm corning cell culture flask ( catalog number 4306 - 41 ) . epimastigotes of t. cruzi cl - brener strain were grown in liver infusion tryptose medium ( lyt ) containing 10% fbs and hemin ( 25 g / ml ) at 28c . fifty percent confluent fibroblasts were infected with 2 10 cl - brener mid - log - phase epimastigotes per ml of hgdmem ( 15 ml ) supplemented with 2% fbs to get a parasite - host cell ratio of 10 : 1 . the nih 3t3 monolayers were washed every 2 days with hgdmem medium until nonadherent parasites were removed , and then fresh hgdmem plus 2% fbs was added . the parasites released from the early infections corresponding to the first 6 days after the first liberation were harvested every day at 2000 g for 5 minutes . the trypomastigotes were separated from amastigotes by immunoprecipitation using 500 l of hybridoma supernatant of monoclonal antibody 2c2b6 , which is specific for the ssp4 surface antigen of amastigotes , per 3 10 parasites at 37c for 1 hour . the trypomastigotes were separated from amastigotes at 800 rpm for 10 seconds in a microcentrifuge and were washed with 1x pbs as many times as necessary to obtain pure parasites . the amastigotes were found in the pellet , and the trypomastigotes were found in the supernatant . purified tissue culture - derived trypomastigotes ( 5 10 ) were harvested at 500 g for 10 minutes and were transferred to 100 20 mm untreated petri dishes containing 1 ml per experiment of hgdmem at different phs ( 4 , 5 , 6 , 7 , 8 , and 9 ) without or with different fbs concentrations ( 0.5% , 1% , 1.5% , and 2% ) incubated at 37c in a 5% co2 atmosphere . after 1 , 2 , 3 , 4 , 5 , 6 , 12 , 24 , 48 , or 72 hours , the parasites were washed , fixed with 4% paraformaldehyde in 1xpbs , washed again , and dropped ( 3 10 parasites/10 l ) over poly - l - lysine - treated slides ( silane - prep slides sigma diagnostics , catalog number s4651 ) inside circles delimited with a liquid - repellent slide marker pen ( electron microscopy sciences catalog number 71310 ) . after the slides were air dried , the parasites were stained with hema 3 according to the protocol supplied by the manufacturer ( fisher diagnostic 840021 ) , and the percentages of trypomastigotes , ifs , and amastigotes were determined for 100 cells analyzed randomly under an optic microscope . trypomastigotes , amastigotes , epimastigotes , or ifs ( 2 10 parasites ) were incubated with 500 l of fresh human serum at 37c for 30 , 60 , and 120 minutes . parasite lysis was determined by adding one volume of trypan blue ( 0.4% in 1x pbs , hycel 23850 ) and counting the complement - resistant parasites in a hemocytometer under a light microscope . three independent experiments , repeated in triplicate each time , were carried out using serum from different donors . the parasites were harvested , washed , fixed , and dropped over poly - l - lysine - treated slides as described above . the parasites were permeabilized with 0.1% triton - x100 for 5 minutes , rinsed extensively with 1x pbs , and treated with 2% bsa ( albumin , bovine , cohn fraction v / 1x pbs ph 7 ) at 37c for 30 minutes . these preparations were washed with pbs and incubated 1 hour at 37c with hybridoma supernatant ( 1 : 50 ) of the amastigote - specific monoclonal antibody 2c2b6 . after rinsing , a secondary antimouse antibody tagged with alexa fluor 488 green diluted 1 : 1000 was layered on the parasites and incubated for 1 hour at 37c . h-1000 ) , and observed under an olympus fluorescence microscope ( bx41 ) equipped with a 60x/1.25 oil iris ph3 uplanfl n objective . the images were captured using an evolution vf fast cooled color media cybernetics camera and were analyzed using the image - pro plus v 6.0 media cybernetics program . the parasites were processed and analyzed as per the indirect immunofluorescence protocol described above , with some specifications . after treatment with a polyclonal antibody against the epimastigotes ( 1 : 200 ) obtained from the mouse and the antimouse antibody tagged with alexa fluor 546 red ( 1 : 1000 ) as the first and second antibodies , the parasites were stained with 10 g / ml dapi ( molecular probes ) . as mentioned before , conditions such as temperature , nutritional environment , and ph stimulate the in vitro differentiation of trypomastigotes into amastigotes [ 58 ] . to determine the best conditions for obtaining the highest rates of transformation , in vitro experiments were performed using tissue - derived trypomastigotes incubated at 37c in hgdmem medium at different phs ( 4 , 5 , 6 , 7 , 8 , and 9 ) , without or with 0.5% , 1% , 1.5% , or 2% fbs for different lengths of time , as described in the materials and methods . the movement and morphology of the parasites were analyzed in fresh preparations under a light microscope , and the relative percentages of trypomastigotes , ifs , and amastigotes were determined . in all of the conditions in which parasite differentiation was achieved , the trypomastigotes showed gradual morphological changes and size reduction ; they transformed from the typical s- or c - shaped bloodstream morphology to diverse ifs that finally reached the rounded or oval shape characteristic of amastigotes after 12 hours of incubation . a progressive reduction of parasite movement concomitant with the gradual shortening of the undulating membrane and flagellum was also observed . although similar morphological patterns of differentiation could be seen in some of the different conditions tested , very different rates of transformation were obtained . incubation at 37c in hgdmem at ph 5 without fbs induced the most efficient rates of differentiation . under these conditions , the level of tissue - derived trypomastigotes decreased concomitantly with increasing amounts of ifs , which reached 85% within 3 hours of incubation ( figure 1 ) . subsequently , as the percentage of ifs decreased , the relative amount of amastigotes increased , and the transformation process was completed after 12 hours of incubation . a homogenous population ( 100% ) of round - shaped forms , which displayed either no flagella or very short protruding flagella and a noticeable reduction of movement , was observed at this point . on the contrary , when tissue - derived trypomastigotes were incubated in hgdmem at ph 4 without fbs , only 30% of the cells were transformed into amastigotes after 3 hours and 65% after 12 hours ( data not shown ) . in addition , only 21% of the trypomastigotes differentiated into amastigotes after 24 hours of incubation at ph 6 without fbs ( data not shown ) . finally , at phs 7 , 8 , and 9 in the absence of fbs as well as those same phs with 0.5% , 1% , 1.5% , and 2% these results indicate that the differentiation process during the in vitro secondary amastigogenesis involves several ifs as a result of the morphological change of the parasites . in addition , the optimal conditions to induce the highest rates of transformation from tissue - derived trypomastigotes into amastigotes were ph 5 , serum - deprivation and 37c . the different developmental forms of t. cruzi can be identified by morphological and biological parasite features , such as the size and shape of the parasite , undulating membrane and flagellum , the position of flagellum emergence , and the size , shape , and relative position of the nucleus and the kinetoplast . the amastigote has a spherical- to oval - shaped form that is 2 to 4 m in diameter with no or very short free flagellum and a kinetoplast located in the middle of the parasite body close to the nucleus . the trypomastigotes have a flagellum and an undulating membrane that extends the entire length of the parasite ( 1625 m long and 2 m diameter ) . trypomastigotes found in the blood of infected mammals have a slender s- or c - shaped form , while the trypomastigotes found in infected insects usually have a straight form . once the best conditions to induce the transformation of the parasite were determined , a detailed morphological analysis was carried out on fixed and stained preparations , and the relative amounts of trypomastigotes , amastigotes , and ifs were determined at different points of the differentiation kinetics at 37c using hgdmem at ph 5 with serum - deprivation ( figure 2 ) . this methodology allowed us to perform a morphometrical analysis and a more precise quantification of the transformation process compared to those obtained with fresh preparations . before initiating the differentiation process , tissue - derived trypomastigotes showed the typical morphology previously described ( figure 2a ) . during the transformation process , the parasite progressively reduced its entire length by 22% at 1 hour , 23% at 2 hours , 39% at 3 hours , 48% at 4 hours , 50% at 5 hours , and 53% at 6 hours of transformation as shown in figure 2 and table 1 . the quantitative analysis indicated that as little as 1 hour of transformation time was enough to induce high rates of ifs ( 80% ) in which the parasites displayed a widening and shortening of their body , flagellum , and undulating membrane ; however , this last one maintained the subterminal emergence site that is characteristic of trypomastigotes ( figure 2b and table 1 ) . after 2 hours of transformation , no trypomastigotes were detected , which indicates that the culture contained 100% ifs . these ifs showed size reduction , a light oval - shaped form , shortening of their flagellum , and an undulating membrane in which their emergence site was slightly displaced to the middle of the parasite body ( figure 2(c ) and table 1 ) . after 3 hours , the morphology of the ifs was very close to the one observed at 2 hours of transformation , but their size continued to reduce and the undulating membrane was not appreciable ( figure 2(d ) and table 1 ) . after 4 and 5 hours of transformation , the ifs showed similar size and morphology to the amastigotes and were only distinguished by the progressive shortening of their flagellum ( figures 2(e ) and 2(f ) and table 1 ) . finally , after 6 hours of transformation , the parasites reached the spherical- to oval - shaped form with no or very short free flagellum ( figure 2(g ) ) , and their morphology was indistinguishable from the one observed in the cell culture - derived amastigotes ( figure 2(h ) and table 1 ) . to assess the reversibility of the differentiation process , the ifs were cultured again in hgdmem at ph 7.2 complemented with 2% fbs after 1 , 2 , 3 , 4 , 5 , 6 , 12 , 24 , 48 or 72 hours of differentiation at 37c using hgdmem at ph 5 with serum deprivation . in all cases , the ifs continued their differentiation , thus indicating that once detonated , this process can not be stopped and is not reversible ( data not shown ) . these results showed that , under the in vitro conditions used , the trypomastigotes differentiated very efficiently into highly homogeneous and pure if populations after 2 hours of induction . to continue with the morphological analysis of the ifs , the shape and position of the nucleus and the kinetoplast at each point of the transformation kinetics were determined . the parasites were then stained with a polyclonal antibody against epimastigote total proteins , which had been previously shown to recognize all of the developmental forms and ifs of t. cruzi . the reactions were developed with an alexa fluor 546 red - conjugated antimouse antibody , and the kinetoplast and nucleus were counterstained with dapi . as shown in previous experiments , the same morphological transformation pattern of the parasite body was found with this technique ( compare figures 2 and 3 ) . nucleus remodeling and a progressive and continuous displacement of the kinetoplast from the posterior to the anterior position with respect to the parasite nucleus were also observed . as shown in table 1 , the distance between the nucleus and the kinetoplast became shorter during the transformation kinetics . at 1 hour of transformation ( figure 3(b ) ) , the distance between both organelles was reduced by 28% ( table 1 ) , even though the ifs showed a subterminal kinetoplast posterior to the nucleus , as commonly observed in trypomastigotes ( figure 3(a ) ) . after 2 hours of transformation , the ifs showed a displacement of their kinetoplast to the middle of the parasite body and reduced the distance with respect to the nucleus ( now remodeled from the lengthened - shaped form to an oval - shaped form ) by 58% ( figure 3(c ) and table 1 ) . after 3 hours of transformation , the kinetoplast remained located posterior to the nucleus , close to the middle part of the parasite body , with a 66% reduction in the distance between both organelles ( figure 3(d ) and table 1 ) . at 4 hours of transformation , the kinetoplast was positioned next to and parallel to the nucleus in a transversal position in the middle of the parasite body and showed 85% reduction in the organelle distance ( figure 3(e ) and table 1 ) . after 5 hours of transformation , the kinetoplast moved to an anterior position with respect to the nucleus , in the middle part of the parasite body and showed a 90% reduction in the distance between the organelles ( figure 3(f ) and table 1 ) . finally , at 6 hours of transformation ( figure 3(g ) and table 1 ) , the parasite morphology and the position of the kinetoplast were very similar to those observed in the tissue - derived amastigotes ( figure 3(h ) and table 1 ) . these results showed that , concomitant with the morphological parasite shape transformation , nucleus remodeling and kinetoplast displacement are observed during the transformation kinetics under the in vitro conditions used . the morphological analysis described above showed homogenous if populations ; nevertheless , the nucleus remodeling and kinetoplast displacement studies indicated that each point of the transformation kinetics is composed of 3 different ifs . the percentages of trypomastigotes , amastigotes and the different ifs were then determined for each time point evaluated in the previous experiment . as shown in figure 4 , 3 different ifs were observed at 1 , 2 , 3 , 4 and 5 hours of in vitro differentiation ( figures 4(a ) and 4(c ) ) . at 1 hour of transformation the first if , which was arbitrarily named if1 , showed similar morphology to the immediate previous parasite stage ( trypomastigotes for the first hour and if3 for 2 , 3 , 4 , and 5 hours ) and represented 2% to 3% of the total parasites . the second intermediary form , named if2 , showed a slightly , but clearly , more differentiated morphology than if1 and represented the most abundant parasite population ( 85% to 90% ) . the third if , still more differentiated than if2 and named if3 ( 2% to 15% ) , displayed characteristics similar to the if1 that emerged from the next time of transformation . at 1 hour of transformation , trypomastigotes comprised only 2% of the culture , which confirms that the transformation process is very efficient under the established conditions ( figures 4(a ) , 4(b ) and 4(c ) ) . moreover , from 1 to 5 hours of transformation were not observed any amastigotes ( panel a ) . finally , at 6 hours of transformation , 100% of the parasites showed a very similar morphology to that of tissue - derived amastigotes ( figures 3(g ) and 3(h ) , table 1 and figures 4(a ) , 4(b ) and 4(c ) ) . the only forms detected after 2 , 3 , 4 and 5 hours of transformation were ifs with a gradually increasing degree of differentiation . therefore , this experimental model could be useful for future analysis of the differential expression between the ifs and the completely differentiated forms of t. cruzi . in addition , even when the ifs at 1 hour of transformation showed morphometric characteristics that corresponded with an if , the presence of contaminant trypomastigotes ( 2% ) rendered this population not useful for differential expression studies ( figure 4 ) . the previous results demonstrated that different ifs , which can be identified by specific morphological characteristics , are generated during amastigogenesis . since it was necessary to know more about their biological properties , a molecular marker was evaluated for the different ifs during the transformation kinetics . it has been reported that the morphological differentiation of the ifs implies the remodeling of parasite surface molecules [ 3 , 8 ] . the expression of the amastigote - specific surface glycoprotein ssp4 was evaluated by indirect immunofluorescence in the diverse ifs as well as in trypomastigotes and amastigotes used as controls . the results presented in figure 5 showed that , as expected , the trypomastigotes did not express the amastigote - specific surface glycoprotein ( figures 5(a ) and 5(b ) ) , while amastigotes displayed heterogeneous levels of ssp4 ( figures 5(o ) and 5(p ) ) . moreover , while the ifs generated at 1 , 2 , and , 3 hours of transformation did not express ssp4 ( from figures 5(c ) to 5(h ) ) , approximately 8% and 36% of parasites at 4 ( figures 5(i ) and 5(j ) ) and 5 hours of differentiation ( figures 5(k ) and 5(l ) ) , respectively , showed low expression levels . in addition , after 6 hours of transformation ( figures 5(m ) and 5(n ) ) , approximately 76% of the parasites showed variable and lower levels of ssp4 compared to culture - derived amastigotes ( figures 5(o ) and 5(p ) ) . finally , after 72 hours of transformation , all parasites showed equivalent expression of ssp4 to that of culture - derived amastigotes ( data not show ) . these results indicated that the consecutive and gradual expression of ssp4 was concomitant with the morphological transformation of trypomastigotes into amastigotes , in which the ifs with a similar morphology to amastigotes were the ones that started expressing the amastigote - specific glycoprotein . moreover , even though the morphology of the parasites at 6 hours of transformation was similar to the tissue - derived amastigote morphology , the expression of this molecular marker indicated that these parasites were not completely transformed at the molecular level . continuing with the biological analysis of the different ifs , their resistance to complement - mediated lysis was determined . as the ifs are obtained during trypomastigote differentiation into amastigotes and both developmental stages are resistant to complement - mediated lysis , it would not be surprising if the ifs are also resistant . however , it was important to evaluate this phenotype because ( 1 ) the ifs are usually intracellular and therefore not exposed to the blood and extracellular space in the vertebrate host and so they would not need to be resistant to complement - mediated lysis and ( 2 ) the ifs obtained during metacyclogenesis and amastigogenesis show very similar morphological characteristics , and very little is known about biological properties that could be used to distinguish them . however , this biological property has not been evaluated in the ifs obtained during amastigogenesis . therefore , the complement - sensitivity of the ifs obtained was evaluated using fresh human serum as described in the materials and methods . as shown in figure 6 , the if at 3 hours of transformation , similar to trypomastigotes and amastigotes moreover , all of the ifs obtained through the transformation kinetics were also resistant to complement - mediated lysis ( data not shown ) . as expected , epimastigotes were sensitive to complement , thus reaching 100% lysed parasites in the first 30 minutes of treatment . these results suggest that the ifs obtained during the in vitro amastigogenesis do not lose the ability to resist complement - mediated lysis . therefore , even if the ifs from metacyclogenesis and amastigogenesis are morphologically similar , they have different biological properties that can be used to distinguish them . the lack of effective in vitro conditions to obtain high rates of synchronic and pure ifs has delayed the understanding of the molecular mechanisms that mediate the cellular remodeling and the identification of molecular markers that are important for amastigogenesis . in the present work , we established an improved method to obtain high rates of pure ifs and correlated parasite differentiation with the expression of a specific developmental marker , complement - mediated lysis sensitivity and morphometrical analysis of the nucleus , kinetoplast and parasite body throughout the complete transformation process . the efficient transformation of cell culture - derived trypomastigotes to amastigotes was induced using an acidic ph and serum deprivation at 37c ( figure 1 ) . the parasites showed dramatic cell restructuring with a gradual and progressive reduction of their size and the readsorption of their flagellum and undulating membrane until they reached the typical round shape of tissue - derived amastigotes ( figure 2 ) . these results are consistent with previous reports that document the existence of several intermediate forms during the life cycle of t. cruzi , either under in vivo or in vitro conditions that showed the same gradual and progressive parasite morphometrical transformation , apparently as an obligatory step for the differentiation process [ 1 , 3 , 5 , 8 , 1822 ] . the quantitative analysis demonstrated that parasite transformation was initiated immediately after the exposure to hgdmem at ph5 and 37c , thus reaching 100% ifs after only 2 hours of incubation ( figure 4 ) ; this indicates a faster and more efficient differentiation rate than previously reported [ 5 , 8 ] . these differences could be the result of the absence of bsa in the differentiation medium used in this study in comparison with the 0.4% bsa contained in the previously reported medium [ 5 , 8 ] . in addition , the condition of the cell culture - derived trypomastigotes used to initiate parasite differentiation may play an important role in the process . the parasites used in all of the experiments in this study were early released trypomastigotes that were obtained from early infections corresponding to the first 6 days after the first trypomastigote liberation , because the parasites released later showed variable , not synchronous and inefficient transformation rates . interestingly , andrews et al , and tomlinson et al , also used only trypomastigotes early released from infected cells for their transformation experiments . even when these authors did not justify this specification the morphological restructuring of t. cruzi during amastigogenesis has shown that the transition from the slender to the spherical or oval parasite shape comprises an extensive remodeling of cellular architecture , the flagellum and the undulating membrane , and a dramatic change in the overall parasite size , apparently as a result of the reorganization of their subpellicular microtubules . this dramatic restructuring also affects the location and morphology of the parasite organelles during t. cruzi differentiation . the kinetoplast of the trypomastigotes is spherical ; whereas in amastigotes , it is elongated [ 8 , 21 , 23 , 24 ] . in contrast , the nuclei of the trypomastigotes are elongated , apparently following the parasite 's shape ; meanwhile , it is round in amastigotes . these morphometrical characteristics of the nucleus and the kinetoplast have been reported to allow the distinction of the t. cruzi developmental stages . therefore , this was the criteria used to initiate the characterization of the ifs obtained ( figure 3 and table 1 ) . the results showed that the morphometrical analysis of the nucleus and the kinetoplast allows the clear identification of the different ifs . the trypomastigotes showed a bigger distance between the nucleus and the kinetoplast than did the amastigotes . in addition , during amastigogenesis , together with the parasite shape change , we observed nucleus remodeling and a progressive and continuous displacement of the kinetoplast from the posterior to the anterior position with respect to the parasite nucleus . similar observations were reported by contreras and collaborators in 2002 , during primary amastigogenesis . as in the quantitative analysis conducted during the morphometrical characterization of the ifs ( figure 2 and table 1 ) , the shape and position of the nucleus and the kinetoplast ( figure 3 and table 1 ) allowed us to very precisely determine that one of these populations was highly predominant even when three different if populations were present at each time of the transformation kinetics ( from 85% to 90% ) , which confirmed a high efficiency of the homogeneity and synchronicity of the differentiation process in our in vitro conditions ( figure 4 ) . differentiation of the diverse developmental stages of t. cruzi has been associated with changes in their biological properties and with the expression of stage - specific surface molecules . moreover , the morphological transformation during primary amastigogenesis has been associated with changes in the glycoprotein profile related to the expression of trans - sialidase and mucin previously described in t. cruzi . the same behavior was observed in this work during the secondary amastigogenesis : contrary to the positive expression of ssp4 observed in the amastigote surface , the trypomastigotes , epimastigotes and ifs at 1 , 2 and 3 hours of transformation did not express this amastigote - specific glycoprotein ( gp ) ( figure 5 ) . in the case of the ifs obtained at 4 , 5 and 6 hours of transformation , low and heterogeneous levels of ssp4 expression were observed . at these times , the total number of parasites expressing ssp4 as well as the expression level of this gp on their surfaces , increased in parallel with the degree of transformation from trypomastigotes to amastigotes , which confirms that the progressive expression of ssp4 can be useful as a molecular marker of differentiation [ 3 , 8 , 21 ] . to initiate and establish infection in the mammalian host , t. cruzi needs to develop several strategies to avoid the immune response . to survive and disseminate in the bloodstream and in the extracellular space , trypomastigotes and amastigotes have developed a stage - specific capacity to resist complement - mediated lysis [ 2527 ] , apparently using stage - specific regulators [ 2830 ] . conversely , epimastigotes found in the insect vector activate the complement cascade , but they are not able to inhibit its lytic action . complement - mediated lysis of the ifs obtained during in vitro secondary amastigogenesis was evaluated . as expected , trypomastigotes and amastigotes , but not epimastigotes , were resistant to the lytic effects of complement . besides this , the ifs obtained during in vitro amastigogenesis were not sensitive to complement lysis ( figure 6 ) . this result indicates that these ifs express the proteins that participate in the stage - specific inhibition of complement activation . nevertheless , future studies will be necessary to elucidate which stage - specific proteins from trypomastigotes and/or amastigotes are expressed by the ifs in order to inhibit complement activation . it has been reported that the ifs generated during the transformation from epimastigote to trypomastigote are sensitive to complement lysis . the ifs obtained during the metacyclogenesis and amastigogenesis are morphologically indistinguishable , which is remarkable , because this biological property might be used as a criterion to distinguish both types of ifs . moreover , this result further supports the existence of the alternative parasite life subcycle that was previously proposed and the hypothesis that this could be the result of the premature lysis of the infected cells or the extracellular parasite differentiation . in summary , we have shown that the experimental differentiation model reported in this work provides , for the first time , a highly synchronous system that could be useful for studying the molecular mechanism of the transformation process and the identification of the regulators involved in differentiation control , with the potential to identify routes to block the cycle of t. cruzi infection .

objective : the objective of this study was to evaluate the dissolution effectiveness of eucalyptus oil , orange oil , xylene , and distilled water on three different endodontic sealers.materials and methods : about 240 samples of root canal sealers ( eighty for each sealer ) were prepared and divided into four groups of 20 each for immersion in different organic solvents . each group was further subdivided into two subgroups ( n = 10 ) for 2 and 10 min of immersion time . the mean percentage of weight loss was determined for each sealer in each solvent at both time periods . data were statistically analyzed by two factor analysis of variance and significance of mean difference was obtained by tukey 's post hoc test ( p < 0.05).results : the lowest level of solubility was observed for adseal followed by apexit plus and endomethasone n at both time periods in all solvents . apexit plus showed no significant ( p > 0.05 ) difference in its dissolution in all the organic solvents except distilled water at both the time periods . the solubility profile of endomethasone n and adseal did not differ significantly among eucalyptus oil , orange oil , and xylene at 2 min and between eucalyptus oil and orange oil at 10 min . however , at 10 min , endomethasone n and adseal showed a more pronounced solubility in xylene as compared to both eucalyptus oil and orange oil.conclusions:in general , xylene was the most effective in dissolving root canal sealers than other organic solvents . essential oils ( eucalyptus oil and orange oil ) were found similar in their ability to dissolve apexit plus and endomethasone n. trypanosoma cruzi undergoes a biphasic life cycle that consists of four alternate developmental stages . in vitro conditions to obtain a synchronic transformation and efficient rates of pure intermediate forms ( ifs ) , which are indispensable for further biochemical , biological , and molecular studies , have not been reported . in the present study , we established an improved method to obtain ifs from secondary amastigogenesis . during the transformation kinetics , we observed progressive decreases in the size of the parasite body , undulating membrane and flagellum that were concomitant with nucleus remodeling and kinetoplast displacement . in addition , a gradual reduction in parasite movement and acquisition of the amastigote - specific ssp4 antigen were observed . therefore , our results showed that the in vitro conditions used obtained large quantities of highly synchronous and pure ifs that were clearly distinguished by morphometrical and molecular analyses . obtaining these ifs represents the first step towards an understanding of the molecular mechanisms involved in amastigogenesis .

there is an increasing recognition of comorbidity between intellectual disability ( i d ) and mental health problems , emotional disorders , and deficits in socialization skills.13 literature reveals that the aging process commences younger in people with i d at approximately 4050 years of age.4 as the previously mentioned problems may further lead to long - term health conditions and mortality,5 effective psychosocial interventions need to be developed for older adults with i d in addition to the pharmacological treatment . life story work ( lsw ) is : the construction , or reconstruction of an individual s life - story and involves the integration of the individual s internal process , as well as the relationships and values with the family , community , and culture in which the individual has developed.6 it creates an opportunity for the person to tell others about their past experiences and then use this life story to benefit them in their present situation.7,8 a variety of forms ( book , digital video disc , or collection of personal items ) can be used , and the content may contain photographs , written biographies , drawings , art pieces , and other aids for understanding a person s own memorable experiences . the lsw program ( p ) in particular , life story book was originally applied to children who were under adoption and foster care services.9 it helped children to develop a sense of identity and continuity in the new setting . lsw was then modified and introduced to a variety of different settings , including children with prior exposure to trauma , people with long - term illnesses,10 older people with or without dementia,11 and recently people with intellectual disabilities.12,13 our past experience and relationships shape our identity and make us who are today.14 this also applies to people with i d and lswp as a way of keeping their past history alive . there is evidence that lswp may encourage people with i d to present and express themselves13 and , hence , improve communication and relationships.15,16 therefore , lswp is like a bridge to create connections among clients , family caregivers , and support workers.17,18 research also indicates that lswp has a positive impact on participants mood . this is especially important for older adults with i d , who are at an increased risk of having mood disorders , particularly depression.19 similar to other aging people , older adults with i d carry with them many unpleasant past life events and face a great deal of unexpected changes , which they may not be able to cope with.20,21 they also have more difficulties in developing and maintaining a stable social support network,22 which is an essential element in fighting mood problems.23 older adults with i d are predisposed to have reduced subjective well - being , given their physical disabilities and related psychological stress.24 however , with a strengthened sense of identity , improved social interaction , and increased pleasure and enjoyment about life , it is reasonable to expect that lswp may enhance the participants quality of life ( qol ) by engaging them in an appropriate level of activities and contacts.2527 hence , it is hypothesized that lswp , when applied to older adults with i d , will have a positive impact on their mood , socialization , and ultimately their qol . thus , the evidence on the use of lswp is immature , and there is a great need for quantitative research evidence to substantiate the potential value of lsw and other cognitive behavioral treatments for patients with id,6,8,28 especially for people with id.16 in addition , to our knowledge , there are very few lswp studies on older adults with i d that have a well - established protocol tailor - made to them , despite their marked disparity from the norm . to address these issues , the present study aimed to develop a training protocol of lswp especially designed for older adults with mild - to - moderate levels of i d and to evaluate the effectiveness of lsw in this group of older adults on enhancing mood , socialization , and qol from a quantitative perspective participants were recruited from nine hostels , day service centers , and sheltered workshops of the hong chi association . the inclusion criteria were : 1 ) > 50 years of age ; 2 ) mild - to - moderate grade i d , according to the service admission record in their personal case files ; and 3 ) without severe psychiatric disorder or behavioral problem . it is suggested that gaining person - centered consent from such participants.29 this was achieved in this study via an ongoing evaluation of both the verbal and nonverbal cues of the participant during the research process , alongside written consent obtained from the guardian of the participant . only participants with both their guardians written informed consent and those who agreed to participate in the program joined the study . this study complied with the ethical standard stipulated by the survey and behavioural research ethics committee of the chinese university of hong kong and ethical approval was granted prior to the data collection . participants were assigned to either the intervention group ( n=32 ) or the control group ( n=28 ) . the intervention group received the lswp intervention led by trained lswp instructors , in addition to their usual daily activities for approximately 6 months . the control group received their usual activities ( ie , training on self - care and daily living skills , basic work skill training , activities that developed their hobbies and interests , physical exercises to maintain or strengthen their physical fitness , vocational training in a sheltered environment ) during this period . assessments on the participants mood , socialization , and qol were conducted at baseline and immediately after the intervention . the protocol of lswp for older people without i d could not fit the special needs of population with i d . therefore , a group of clinical psychologists developed a life story book protocol which was designed specifically for this population . in developing this protocol , the following recommendations were taken into consideration : 1 ) the effective features of a life story intervention design for nursing home residents ; and 2 ) suggestions on the best format for compiling life history resources for older people living in institutional settings.16,30 this life story book protocol consisted of 16 structured one - to - one or group sessions , each lasting for 1.52 hours . the entire program spanned a period of approximately 6 months . it comprised a series of activities of various psychosocial elements , such as field visits , outings , production of life story book with photos , presentation , group sharing , and collecting feedback from the caregiver . lswp instructors work with participants and their family members to collect information and photos that tell the participants life stories . lswp instructors also help participants to express their feelings on their life stories in a caring and accepting atmosphere . participants are encouraged to use the life story books to share their life stories and achievements with other people . participants were guided by trained lswp instructors , who were experienced tutors for people with i d and received training and regular supervision from the clinical psychologists . the protocol provided step - by - step guidelines for the instructors to produce an individualized life story book for each client . the life story book enabled clients to gather current information about themselves ( the present ) and their history ( the past ) . moreover , it was a useful tool that could assist the clients to express themselves and tell their own story to people around them . , each participant will have a personalized life story book that includes the recording of significant people , places , and events for themselves ( table 1 ) . participants were recruited from nine hostels , day service centers , and sheltered workshops of the hong chi association . the inclusion criteria were : 1 ) > 50 years of age ; 2 ) mild - to - moderate grade i d , according to the service admission record in their personal case files ; and 3 ) without severe psychiatric disorder or behavioral problem . it is suggested that gaining person - centered consent from such participants.29 this was achieved in this study via an ongoing evaluation of both the verbal and nonverbal cues of the participant during the research process , alongside written consent obtained from the guardian of the participant . only participants with both their guardians written informed consent and those who agreed to participate in the program joined the study . this study complied with the ethical standard stipulated by the survey and behavioural research ethics committee of the chinese university of hong kong and ethical approval was granted prior to the data collection . participants were assigned to either the intervention group ( n=32 ) or the control group ( n=28 ) . the intervention group received the lswp intervention led by trained lswp instructors , in addition to their usual daily activities for approximately 6 months . the control group received their usual activities ( ie , training on self - care and daily living skills , basic work skill training , activities that developed their hobbies and interests , physical exercises to maintain or strengthen their physical fitness , vocational training in a sheltered environment ) during this period . assessments on the participants mood , socialization , and qol were conducted at baseline and immediately after the intervention . the protocol of lswp for older people without i d could not fit the special needs of population with i d . therefore , a group of clinical psychologists developed a life story book protocol which was designed specifically for this population . in developing this protocol , the following recommendations were taken into consideration : 1 ) the effective features of a life story intervention design for nursing home residents ; and 2 ) suggestions on the best format for compiling life history resources for older people living in institutional settings.16,30 this life story book protocol consisted of 16 structured one - to - one or group sessions , each lasting for 1.52 hours . the entire program spanned a period of approximately 6 months . it comprised a series of activities of various psychosocial elements , such as field visits , outings , production of life story book with photos , presentation , group sharing , and collecting feedback from the caregiver . lswp instructors work with participants and their family members to collect information and photos that tell the participants life stories . lswp instructors also help participants to express their feelings on their life stories in a caring and accepting atmosphere . participants are encouraged to use the life story books to share their life stories and achievements with other people . participants were guided by trained lswp instructors , who were experienced tutors for people with i d and received training and regular supervision from the clinical psychologists . the protocol provided step - by - step guidelines for the instructors to produce an individualized life story book for each client . the life story book enabled clients to gather current information about themselves ( the present ) and their history ( the past ) . moreover , it was a useful tool that could assist the clients to express themselves and tell their own story to people around them . , each participant will have a personalized life story book that includes the recording of significant people , places , and events for themselves ( table 1 ) . validated assessment tools were used to collect data for both intervention and control groups at both pretest and posttest periods . as mood , socialization , and qol were the outcomes of interest , corresponding assessment tools of mood interest and pleasure questionnaire ( mipq),31 vineland adaptive behavior scales , second edition ( vineland - ii),32 and personal well - being index intellectual disability ( cantonese ) , third edition ( pwi - id),33 were used respectively . these standardized measurements were chosen as the most appropriate to detect any change in the outcomes of interest for the subjects over the study period . the selected assessment tools demonstrate good reliability and validity . for the purpose of this study , the analysis of vineland - ii mainly focused on the socialization domain , while the communication abilities were perceived as a background and independent variable . data were analyzed using the statistical package for social sciences version 15 ( spss inc . , chicago , il , usa ) . independent samples t - tests ( and chi - square when appropriate ) were used to compare demographic characteristics between the control and intervention groups at baseline . two - way repeated measures of analysis of variance was further used to examine the effectiveness of the lswp . the difference in each function or well - being was calculated by subtracting participants score measured at baseline from their score measured 2 weeks after the intervention . independent samples t - tests were further conducted to compare the direction and magnitude of change between the control and intervention groups . table 2 summarizes the baseline demographics of the participants . a total of 60 participants with i d were recruited into the study and were assigned to either the lswp intervention group ( n=32 ) or control group ( n=28 ) . results showed that there were no significant differences between the control and intervention groups in terms of demographic factors , including age , sex , education level , communication abilities , marital status , and types of services received . similarly , no significant differences were detected between the two groups on baseline measurements of personal well - being , mood , interest , and pleasure or adaptive behavior on socialization , as measured by the pwi , mipq , and vineland - ii scales , respectively . the lswp had the potential to prevent the deterioration of mood ( as measured by mipq ) of older adults with i d ( table 2 ) . results of repeated measures analysis of variance showed a marginally significant main effect of time ( p=0.07 ) , but the main effect of intervention / control group on the mipq score was insignificant ( p=0.85 ) . the overall interaction effect of time and intervention was also found to be marginally significant ( p=0.09 ) . further analysis showed that the mean mipq score declined from 67.3662.27 in the control group ; whereas , the mean mipq score for the intervention group remained almost the same from 65.6265.35 , indicating the effectiveness of lswp in preventing the negative change in mood , interest , and pleasure of the participants . independent samples t - tests were further conducted on the mean difference in the subdomains of mipq between the control and the intervention groups ( table 3 ) . interest and pleasure ( p=0.04 ) , while no differences were evident in other domains . the lswp showed promise in improving the socialization skills ( as measured by vineland - ii ) of older adults with i d ( table 3 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.54 ) or intervention / control group ( p=0.58 ) on the socialization skills . similarly , the overall interaction effect of time and intervention was not significant ( p=0.56 ) . independent samples t - tests were also conducted on the mean difference in adaptive behaviors and other subscales of the vineland - ii scale between the control and the intervention groups , but there were no significant differences . as stated in table 4 , concerning their socialization skills , the intervention group improved a little ( m = 3.19 ; sd = 20.66 ) while their counterparts in the control group remained the same ( m = 0.07 ; sd = 19.32 ) . the lswp enhanced qol ( as measured by the pwi - id ) of older adults with i d ( table 4 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.77 ) or intervention / control group ( p=0.96 ) on the pwi score . but the overall interaction effect of time and intervention was marginally significant ( p=0.09 ) . the mean pwi score declined from 82.04 to 76.24 in the control group while that for the intervention group increased from 75.24 to 81.31 , indicating the lswp tended to enhance the qol among older adults with i d . the mean scores of both the intervention and control groups were higher than that of the norm in hong kong which is 63.99.33 independent samples t - tests were further conducted on the change of mean ( m ) in different pwi life domains between the control and the intervention groups ( table 5 ) . particularly , the personal health status of participants in the intervention group improved after receiving the lsw treatment ( m = 7.60 ) , but it deteriorated in the control group ( m = -8.57 ) . there was marked improvement concerning community - connectedness among the intervention group ( m = 6.20 ; standard deviation [ sd ] = 29.91 ) ; whereas , a decrease in community - connectedness was detected in the control group ( m = -21.43 ; sd = 34.97 ) . the difference between the two groups was statistically significant ( p=0.01 ; table 5 ) . although there were no significant differences in the other five life domains between the control and the intervention group , the outcome of the intervention group seemed to be better than that of the control group in all of these domains , as demonstrated by the mean differences ( table 5 ) . the effectiveness of lsw tended to vary , depending on the participants communication abilities . to examine whether the effectiveness of lswp was dependent on the participants communication abilities , we divided the participants who received the intervention into two groups based on their communication abilities . a mean cut - off score of 27.7 ( according to the vineland - ii manual on adults with moderate - grade mental retardation ) was used to categorize participants into a low communication group ( mean score , 27.7 ) , and a high communication group ( mean score , > 27.7 ) . as a result , 18 participants were assigned to the low communication group , and 14 participants were assigned to the high communication group . independent samples t - tests were conducted on the change of mean ( m ) in different outcome variables between the low and high communication groups . compared to the low communication score group ( m = -0.95 ; sd = 29.06 ) , the lswp seemed to be more effective in improving the pwi of the group with the higher communication score ( m = 10.60 ; sd = 15.89 ) and its subdomains except for personal safety although this did not reach statistical significance . a similar pattern was observed for the socialization skills ( low communication score group , m = 0.44 , sd = 6.30 ; high communication score group , m = 6.71 , sd = 30.69 ) . however , all of these observed differences did not reach statistical significance , partly due to a relatively small sample in our study . table 2 summarizes the baseline demographics of the participants . a total of 60 participants with i d were recruited into the study and were assigned to either the lswp intervention group ( n=32 ) or control group ( n=28 ) . results showed that there were no significant differences between the control and intervention groups in terms of demographic factors , including age , sex , education level , communication abilities , marital status , and types of services received . similarly , no significant differences were detected between the two groups on baseline measurements of personal well - being , mood , interest , and pleasure or adaptive behavior on socialization , as measured by the pwi , mipq , and vineland - ii scales , respectively . the lswp had the potential to prevent the deterioration of mood ( as measured by mipq ) of older adults with i d ( table 2 ) . results of repeated measures analysis of variance showed a marginally significant main effect of time ( p=0.07 ) , but the main effect of intervention / control group on the mipq score was insignificant ( p=0.85 ) . the overall interaction effect of time and intervention was also found to be marginally significant ( p=0.09 ) . further analysis showed that the mean mipq score declined from 67.3662.27 in the control group ; whereas , the mean mipq score for the intervention group remained almost the same from 65.6265.35 , indicating the effectiveness of lswp in preventing the negative change in mood , interest , and pleasure of the participants . independent samples t - tests were further conducted on the mean difference in the subdomains of mipq between the control and the intervention groups ( table 3 ) . interest and pleasure ( p=0.04 ) , while no differences were evident in other domains . the lswp showed promise in improving the socialization skills ( as measured by vineland - ii ) of older adults with i d ( table 3 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.54 ) or intervention / control group ( p=0.58 ) on the socialization skills . similarly , the overall interaction effect of time and intervention was not significant ( p=0.56 ) . independent samples t - tests were also conducted on the mean difference in adaptive behaviors and other subscales of the vineland - ii scale between the control and the intervention groups , but there were no significant differences . as stated in table 4 , concerning their socialization skills , the intervention group improved a little ( m = 3.19 ; sd = 20.66 ) while their counterparts in the control group remained the same ( m = 0.07 ; sd = 19.32 ) . the lswp enhanced qol ( as measured by the pwi - id ) of older adults with i d ( table 4 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.77 ) or intervention / control group ( p=0.96 ) on the pwi score . but the overall interaction effect of time and intervention was marginally significant ( p=0.09 ) . the mean pwi score declined from 82.04 to 76.24 in the control group while that for the intervention group increased from 75.24 to 81.31 , indicating the lswp tended to enhance the qol among older adults with i d . the mean scores of both the intervention and control groups were higher than that of the norm in hong kong which is 63.99.33 independent samples t - tests were further conducted on the change of mean ( m ) in different pwi life domains between the control and the intervention groups ( table 5 ) . particularly , the personal health status of participants in the intervention group improved after receiving the lsw treatment ( m = 7.60 ) , but it deteriorated in the control group ( m = -8.57 ) . there was marked improvement concerning community - connectedness among the intervention group ( m = 6.20 ; standard deviation [ sd ] = 29.91 ) ; whereas , a decrease in community - connectedness was detected in the control group ( m = -21.43 ; sd = 34.97 ) . the difference between the two groups was statistically significant ( p=0.01 ; table 5 ) . although there were no significant differences in the other five life domains between the control and the intervention group , the outcome of the intervention group seemed to be better than that of the control group in all of these domains , as demonstrated by the mean differences ( table 5 ) . the effectiveness of lsw tended to vary , depending on the participants communication abilities . to examine whether the effectiveness of lswp was dependent on the participants communication abilities , we divided the participants who received the intervention into two groups based on their communication abilities . a mean cut - off score of 27.7 ( according to the vineland - ii manual on adults with moderate - grade mental retardation ) was used to categorize participants into a low communication group ( mean score , 27.7 ) , and a high communication group ( mean score , > 27.7 ) . as a result , 18 participants were assigned to the low communication group , and 14 participants were assigned to the high communication group . independent samples t - tests were conducted on the change of mean ( m ) in different outcome variables between the low and high communication groups . compared to the low communication score group ( m = -0.95 ; sd = 29.06 ) , the lswp seemed to be more effective in improving the pwi of the group with the higher communication score ( m = 10.60 ; sd = 15.89 ) and its subdomains except for personal safety although this did not reach statistical significance . a similar pattern was observed for the socialization skills ( low communication score group , m = 0.44 , sd = 6.30 ; high communication score group , m = 6.71 , sd = 30.69 ) . however , all of these observed differences did not reach statistical significance , partly due to a relatively small sample in our study . the present study represents one of the first attempts , using quantitative assessments , to evaluate the effectiveness of lswp in enhancing mood , socialization , and qol in older adults with i d . the results of the study showed that our lswp was generally effective in improving qol . it had the potential to prevent the deterioration of mood and showed promise in improving the socialization skills in older adults with i d . in addition , we also found that the effectiveness of lsw tended to vary , depending on the participants communication abilities . first , consistent with the results of the previous studies , our lswp improved the personal well - being of the participants , and it especially enhanced participants perceived personal health and sense of community - connectedness.8,34 since there is evidence that happiness and life satisfaction prospectively predict self - rated health,35 and people s self - rated health status is significantly and positively correlated with their actual physical and mental health , which may further contribute to better qol , the improvement of the participants perceived personal health was quite a desirable outcome of lswp.36 to some extent , it indicated participants ameliorated health status and qol after receiving lswp . second , although a significant improvement in mood was not detected in the participants of lswp , the results were encouraging in that , compared to the control group who experienced a drop in the mipq score , those who participated in the lswp had a relatively stable mood reflected by the mipq score . the lswp participants were more emotionally stable , showed interest , and actively participated in the lswp . a previous study has shown that emotional competence is positively and significantly associated with happiness and life satisfaction.37 as a matter of fact , it has been shown that people with i d are more likely to suffer from emotional disorders,1,19 and the severity of emotional problems including depressed mood and the loss of interest was positively correlated with age.5,38 these findings partly explain why a natural decline in the mipq score was detected in the control group in the present study . since previous studies have also found that emotional disorders may further affect long - term health conditions and mortality , effective interventions are needed.5 this study discovered the potential of our lswp to act as a protective factor in preventing the deterioration of mood in people with i d . third , results showed a positive trend of improvement among subjects in the intervention group in terms of their socialization skills , although this was not statistically significant . it could be that participants need some time to practice their improved social skills and , therefore , the improvement may not be immediately observable . in the program , it was believed that the tailor - made life story book with photos from lsw could assist the recall of past memories of the participants , help them to express themselves , and promote sharing with others . this aspect of change could be regarded as the improvement in socialization skills and community - connectedness . to some extent , the stronger sense of community - connectedness , the increased interest and pleasure , and the improved socialization skills can be perceived as enhanced participation , which has been found to be a valuable experience for people with different types of disabilities.39 last but not least participants with better communication skills seemed to benefit more from the program . compared to the group with a lower communication score , the lswp seemed to be more effective for the high communication group in improving their socialization skills and qol although statistical significance was not reached . a possible explanation was that those who had better expressive and comprehension skills were more actively involved in the lswp as compared to those with relatively limited verbal abilities . although the current study represents the first attempt to comprehensively and quantitatively evaluate the effectiveness of lswp on older adults with i d , the results might be affected by the relatively small sample size . in addition , as all of the participants were recruited from the same rehabilitation organization and the intervention and control group were not equally distributed in their living conditions for the ease of administration in the delivery of lswp , this might weaken the rigor and generalizability of the findings . therefore , further studies with larger sample sizes are warranted to provide more convincing evidence . . it would be better if regular booster trainings could be provided after the completion of the lsw , so that participants could update their lsw and refresh their memory and learning . the long - term effect of lsw could be further examined if booster trainings were incorporated into the program .

objectivethis study examines the effectiveness of a life story work program ( lswp ) in older adults with mild - to - moderate levels of intellectual disability ( id).methodsusing a quasiexperimental design , this study assigned 60 older adults who were between 5090 years old with mild - to - moderate levels of i d to receive either the lswp ( intervention group , n=32 ) or usual activities ( control group , n=28 ) during a period of 6 months . evaluation was made based on the outcomes assessed by the mood interest and pleasure questionnaire , the vineland adaptive behavior scales , and the personal wellbeing index id.results and conclusionlswp shows potential for improving the quality of life and preventing the loss of interest and pleasure in older adults with i d . it also shows promise in enhancing their socialization skills . patients with better communication abilities seemed to benefit more from the lswp . the russian nickel refineries located in the cities of nikel and zapolyarny close to the norwegian border are responsible for extensive sulfur dioxide and nickel pollution , as well as severe ecological damage in both countries . the aim of our study was to investigate human nickel exposure in the populations living on both sides of the norwegian - russian border . the design was a cross - sectional population - based study of adults aged 18 - 69 years residing in sor - varanger municipality , norway , and nikel and zapolyarny , russia , during 1994 and 1995 . individual exposure to nickel was assessed by measurements of nickel in urine using electrothermal atomic absorption spectrometry . for controls , urine was collected from adults in the russian cities of apatity and umba ( kola peninsula ) and the norwegian city of tromso , all of which are locations without nearby point sources of nickel . altogether 2,233 urine specimens were analysed for nickel . people living in nikel had the highest concentrations ( median 3.4 microg / l ) , followed by umba ( median 2.7 microg / l ) , zapolyarny ( median 2.0 microg / l ) , apatity ( median 1.9 microg / l ) , tromso ( median 1.2 microg / l ) , and sor - varanger ( median 0.6 microg / l ) . regardless of geographical location , the russian study groups all had a higher urinary - nickel average than those in norway ( p<0.001 ) . with the exception of nikel , neither the russian nor the norwegian urinary - nickel levels were associated with residence location near a russian nickel refinery . we concluded that industrial nickel pollution alone could not explain the observed discrepancy between norway and russia ; we also discuss other possible nickel exposure sources that may account for the high urinary levels found in russia.imagesfigure 1figure 2figure 3figure 4figure 5

corticosteroids remain the mainstay of treatment for the vast majority of patients with immune - mediated uveitis . however , those patients with active inflammation who are intolerant of or unresponsive to steroids require therapy with other immunosuppressive agents trying to prevent the potential sequelae associated with this vision - threatening condition . the off - label use of biologic agents and particularly those blocking tumor necrosis factor - alpha ( tnf- ) has demonstrated encouraging results when employed for management of patients with immune - mediated uveitis refractory to conventional treatment since their first reported use in 2001 . potential advantages of these agents when compared with traditional immunosuppressors include a substantial efficacy in recalcitrant cases , as well as a lower total immunosuppressive load , a rapid clinical effect , good safety profile , and significant improvement in quality of life . the present evidence shows that infliximab and adalimumab have the highest level of evidence and grade of recommendation , and thus both may be considered as first - line or second - line immunomodulatory agents for treatment of immune - mediated uveitis depending on which systemic immunologic disorder is associated with the intraocular inflammation [ 6 , 8 ] . golimumab ( glm ) ( trade name simponi ) , a fully human anti - tnf- monoclonal antibody , was approved by the us food and drug administration in 2009 for the use with methotrexate ( mtx ) in adults with moderate - to - severe active rheumatoid arthritis ( ra ) and with or without mtx or other biologic disease - modifying antirheumatic drugs in adults with active psoriatic arthritis ( psa ) or active ankylosing spondylitis ( as ) . some other studies have addressed the potential use of glm for treatment of uveitis , mainly associated with rheumatologic conditions [ 11 , 12 ] . we would like to present the results from three uveitis units in spain when using glm for treatment of patients with immune - mediated uveitis of various etiologies that had been resistant to several immunosuppressive agents . patients with different types of active immune - mediated uveitis that had been resistant to local and systemic corticosteroids and at least one additional immunosuppressive agent and who were treated with glm during at least 6 months were included in this study . table 1 lists demographic and diagnostic information for the 13 patients who form the basis of this report . in 12/13 patients ( 92.3% ) glm was at least the second biologic agent used for treatment of uveitis , whereas glm was used as first - line biologic therapy in one patient ( patient 6 ) . table 2 shows previous treatment regimens employed for management of uveitis in all included patients . we defined primary failure as an absence of a two - step decrease in level of inflammation ( e.g. , anterior chamber and/or vitreous cells ) or a decrease to grade 0 . we classified control of inflammation as grade 0 cells in both anterior and posterior segments in addition to absence of other signs of intraocular inflammation ( cystoid macular edema ( cme ) and vasculitis ) . all included patients received 50 mg of subcutaneous glm every four weeks during at least 6 months without modifications during the follow - up . chest x - ray , tuberculin skin test , and quantiferon - tb gold were performed in all patients before treatment . glm was the only immunomodulatory agent used in six of them . in seven patients , topical steroids were used by three patients ( patients 6 , 8 , and 9 ) at the beginning of the follow - up and were slowly tapered and discontinued after one month in all of them . uveitis clinical evaluation was performed at least four times ( before treatment and 1 , 3 , and 6 months after initiation of therapy with glm ) in all included patients . clinical evaluation included visual acuity ( bcva ; best - corrected snellen va ) and ophthalmic examination . anterior chamber was graded according to the classification established by the standardisation of uveitis nomenclature ; whereas the national eye institute system was adopted for grading vitreous inflammation [ 13 , 14 ] . optical coherence tomography ( cirrus hd - oct , carl zeiss meditec , dublin , ca , usa ) was used before and after treatment in both groups of patients to determine the presence of cme . the 1 mm central retinal thickness was evaluated using the macular cube strategy 512 128 in all patients at each study visit . macular edema was defined as central macular thickness > 300 and/or presence of intraretinal cysts in oct . fluorescein angiogram ( fa ) was performed routinely before and after starting treatment ( between 1 and 3 months after initiation of therapy ) to determine the presence or absence of retinal angiographic leakage . treatment - related side effects were assessed on each visit with a thorough review of systems and complete blood - cell counts , blood urea nitrogen ( bun ) level , creatinine level , and liver function test parameters obtained on an every study visit basis . results were expressed as mean sd for variables with a normal distribution or as median ( 25th75th interquartile range ( iqr ) ) when they were not normally distributed . eight men and 5 women ( 22 affected eyes ) with a median age of 30 years ( range 2038 ) and active immune - mediated uveitides were studied . uveitis was anterior in 8 patients ( 61.5% ) , intermediate in 1 patient ( 7.6% ) , and panuveitis in 4 patients ( 30.7% ) . all included patients ( 13/13 ) received previous treatment with systemic steroids ( using intravenous pulses of methyl - prednisolone ) in two of them . in addition , four patients ( 30.7% ) received coadjuvant intraocular steroids ( 2 intravitreal triamcinolone injections and 2 dexamethasone intravitreal implants ) . about traditional immunosuppressors , all included patients had been treated with methotrexate at any time prior to glm therapy , whereas cyclosporine a was used in 2 ( 15.38% ) , azathioprine in 1 ( 7.6% ) , tacrolimus in 1 ( 7.6% ) , mycophenolate mofetil in 1 ( 7.6% ) , cyclophosphamide in 1 ( 7.6% ) , and leflunomide in 1 ( 7.6% ) . only one patient received glm as first - line biologic therapy , whereas 12 patients received previous treatment with at least one other biologic agent . from the latter , 12 patients received treatment with at least one biologic drug prior to glm , 7 patients ( 53.8% ) received treatment with at least 2 biologics , 3 patients ( 23% ) received treatment with at least 3 biologics , and 1 patient ( 7.6% ) received treatment with at least 4 biologics . infliximab ( ifx ) was used as first biologic agent in 8 patients ( 61.5% ) , adalimumab ( ada ) in 3 patients , ( 23% ) , and etanercept ( etn ) in 1 patient ( 7.6% ) . ada was used as second biologic agent in 6 patients ( 46.1% ) and etn in 1 patient ( 7.6% ) . mean time on second biologic agent was 25.5 months ( range 1557 ) . abatacept was used as third biologic agent in 2 patients ( 15.3% ) and certolizumab in 1 patient ( 7.6% ) . etn was used as fourth biologic agent in 1 patient ( 7.6% ) during 7 months . coadjuvant immunosuppressive therapy was used in 7 of the studied patients including methotrexate ( 4 patients ) , azathioprine ( 1 patient ) , mycophenolate mofetil ( 1 patient ) , and leflunomide ( 1 patient ) . glm therapy achieved complete control of inflammation in 12/13 patients ( 92.3% ) after six months of treatment . the mean bcva increased from a basal value ( before initiation of glm ) of 0.60 to 0.68 at the six - month endpoint ( p = 0.009 ) . only one patient , patient number 12 , showed a score of anterior chamber and/or vitreous inflammation different than zero at the six - month endpoint . the mean 1 mm central retinal thickness decreased from a basal value of 317 to 261,2 at the six - month endpoint ( p = 0.05 ) . figure 1 shows changes in mean values of macular thickness ( 1 mm central thickness ) of all included patients over the study period . there was no evidence of active retinal vasculitis before initiation of therapy in any of the included patients . fluorescein angiogram results did not differ from those observed with oct regarding the presence of cme . only a mild and local cutaneous reaction was recorded in two patients ( patients 5 and 9 ) among all included patients over the entire study period . these results suggest that glm is well tolerated and is associated with control of inflammation in 92,3% of a heterogeneous group of immune - mediated uveitis patients resistant to traditional therapy and other biologic agents . the use of glm is also associated with short - term improvement in mean values of bcva and decrease of mean values of central retinal thickness . despite the evident limitations of this study , including its retrospective design , lack of a control group , short follow - up , and limited number of patients , the results suggest that further evaluation of this modality is appropriate . tnf- is recognized as one of the main inflammatory cytokines involved in the pathogenesis of immune - mediated uveitis [ 1416 ] . this ubiquitous cytokine plays a key role in initiating and maintaining the inflammatory processes by orchestrating leukocyte infiltration , dendritic cell maturation , and macrophage activation and driving t - helper lymphocytes ' response . therapy with two tnf- inhibitors , infliximab and adalimumab , has been proven to be effective for treatment of immune - mediated uveitis with considerable levels of recommendation and evidence [ 6 , 8 ] . the clinical efficacy of other tnf- blockers such as glm needs to be demonstrated and thus this drug is considered only as an alternative to those patients who have failed to respond to first - line tnf- inhibitors . however , it is necessary to emphasize that not all patients respond to their first anti - tnf agent , and so it is clearly useful to have a range of effective therapeutic options to treat those patients with severe and refractory immune - mediated uveitis . in this setting , . a potential limitation of these results could be related to the inclusion of less severe uveitis considering that we include 8 patients with anterior uveitis . however , anterior uveitis in these patients was associated with juvenile idiopathic arthritis ( patients 5 , 9 , 10 , and 11 ) and hla - b27 + haplotype ( patients 1 , 4 , 6 , and 8) . despite recent therapeutic progress , jia - associated uveitis has a severe course and the potential for long - term complications , including blindness . hla - b27 positive - associated anterior uveitis is associated with a substantially higher incidence of ocular complications and has a much worse prognosis when compared with hla - b27 negative - associated anterior uveitis . moreover , some of the patients with anterior uveitis ( patients 2 , 9 , and 11 ) had concomitant macular edema , the most vision - threatening complication associated with uveitis [ 20 , 21 ] . in this the seven cases that were included in our paper were severe and vision - threatening cases refractive to conventional and nonconventional treatment for such conditions . of note , patients included in the present study had a severe inflammatory condition which was resistant to several treatment - regimens inflammatory conditions . the positive response observed in almost all included patients needs to be analysed in this mentioned difficult clinical setting . this inclusion bias in addition to the short follow - up may have influenced the limited improvement in visual acuity observed in our study considering the long and severe course of intraocular inflammation in our patients . tnf- is one of the inflammatory cytokines that upregulates intraocular production of vegf [ 22 , 23 ] , which plays a crucial role in the pathogenesis of cme . we have previously reported how another tnf- blocker , adalimumab , induces a reduction in plasma vegf levels when employed for treatment of immune - mediated uveitis , which may correlate with clinical improvement . in our study glm also demonstrated a beneficial effect on cme thus strengthening the idea of a comparable efficacy with first - line tnf- blockers . previous reports on the use of glm for treatment of uveitis have focused on those patients with uveitis secondary to behet disease , juvenile idiopathic arthritis , and/or hla - b27 + haplotype . to the best of our knowledge , this is the first report of glm employed in the treatment of uveitis associated with sarcoidosis , vogt - koyanagi - harada disease , and/or psoriatic arthritis . the increasing potential treatment indications for glm may be of high interest in the therapeutical decision making of chronic uveitis patients . the main aim of this study is to show these encouraging results on the use of glm for treatment of immune - mediated uveitis . although these results are preliminary , further studies including a higher and more homogeneous group of patients are warranted . the small sizes of the nanoparticles and large surface to volume ratio put the nanoparticles in a position for tremendous and wide applications essentially in biomedicine . though the small sizes of engineered nanoparticles have been linked with highly desirable properties ( mechanical , electrical , and chemical ) for specific uses , yet these same desirable properties are also likely to be associated with unwanted biological / toxicological reactivity . although metal nanoparticles have received increasing attention due to their widespread medical , consumer , industrial , and military applications , studies have correlated particle size of some metal - based nanoparticles ( e.g. , ag , au , and cu ) with toxicity , even if the same material is relatively inert in its bulk form [ 2 , 3 ] . indeed , there are increasing concerns about the safety of nanoparticles for human health and environment [ 46 ] , highlighting the need for further investigations on the safety of metal nanoparticles . moreover , data on the safety / toxicity profiles of metal nanoparticles are scarce . perhaps , the fears of health risks may not be completely unfounded , if the small sizes and the large surface area to volume ratio as well as the chemical reactivity and/or biological activity of the nanoparticles are to be considered . also there are studies revealing the potential of nanoparticles to alter normal physiology by interacting with biomolecules in living cells [ 79 ] thereby causing adverse effects at the cellular , subcellular , and protein levels . furthermore , nanoparticles enter the human body through ingestion , inhalation , and skin contact or genitourinary tract and become deposited in vital organs such as brain , liver , or kidneys [ 10 , 11 ] . studies have shown that nanoparticles may change or damage cellular processes by passing through cellular membranes to interact with biomolecules leading to dna and protein damage [ 12 , 13 ] or cross the blood - brain barrier to cause neurotoxicity . these factors underscore the urgent need for investigations aimed at establishing the influence of nanoparticles on biochemical parameters . the present study determined the effect of the oral administration of silver nanoparticle on some biochemical parameters in wistar rats . alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , and alkaline phosphatase ( alp ) assay kits were supplied by randox laboratory ( crumlin , uk ) . all other reagents were of analytical grade and were prepared in distilled water unless otherwise stated . male rats of wistar strain weighing between 170 and 180 g were obtained from the university of ibadan , ibadan , nigeria . animals were housed in hygienic environment and allowed to acclimatize for a week before commencement of treatments . the preparation and characterization of silver nanoparticles were as previously reported [ 15 , 16 ] . briefly , the agnps were made by adding 100 mm silver nitrate to a 1% ( w / v ) tannic acid solution ( ph adjusted to 8 by adding 150 mm potassium carbonate ) of polyvinylpyrrolidone ( pvp ) with stirring . the agnps were filtered using the 0.22 m filter and characterized by uv / vis spectrophotometry ( biotek epoch , usa ) , inductively coupled plasma optical emission spectrometry ( icp - oes , cambridge , united kingdom ) , and transmission electron microscopy ( tem , brno , czech republic ) . groups b , c , and d each were daily administered with 100 , 1000 , and 5000 mg / kg agnps , respectively , for 7 days . groups e , f , and g each were daily administered with 100 , 1000 , and 5000 mg / kg agnps , respectively , for 14 days . groups b , c , and d each were daily administered with 100 , 1000 , and 5000 mg / kg agnps , respectively , for 7 days . groups e , f , and g each were daily administered with 100 , 1000 , and 5000 mg / kg agnps , respectively , for 14 days . doses were fixed based on a recent report which demonstrated that the ld50 for silver nanoparticles ( agnps ) was greater than 5000 mg / kg . all treatments were orally administered with the aid of a cannula and animals sacrificed alternately 24 hours after cessation of treatments . the study was carried out in accordance with institutional guidelines on the handling of animals as approved for scientific research . 24 hours following cessation of last treatments , rats were sacrificed under anaesthetization in slight chloroform . blood samples were obtained by cardiac puncture into clean centrifuge bottles and spun at 4000 g for 5 minutes ( heraeus labofuge 300 , thermo scientific , hampshire , uk ) to yield the serum . the heart , liver , and kidneys from each animal were excised and weighed immediately . excised tissues were homogenized in ice - cold 0.25 m sucrose ( 1 : 5 w / v ) using a teflon homogeniser ( sigma - aldrich chemie gmbh , munich , germany ) . the tissue homogenates were centrifuged at 4000 g for 5 minutes ( heraeus labofuge 300 , thermo scientific , hampshire , uk ) to remove unbroken particulates . the following biochemical parameters were determined in rat serum , kidney , liver , and heart using a uv / vis spectrophotometer ( shimadzu , kyoto , japan ) where applicable ; protein concentrations in serum and tissue homogenates were estimated as described by gornall et al . . aspartate aminotransferase ( ast ) was determined using commercial randox kit [ 19 , 20 ] . data were subjected to statistical analysis using one - way anova with graphpad prism 3.0 ( graphpad software inc . , san diego , ca ) . the agnps had a maximal uv absorption at 430 nm with diameter sizes ranging between 8 and 20 nm ( figure 1 ) . treatment with agnps at the dose of 5000 mg / kg caused dullness in the rats , with one death recorded in the same group . the relative organ - to - body weight ratios for the animals are presented in table 1 . there are no significant differences when comparing between the agnps treatment groups and the control . the weights of the rats treated with silver nanoparticles increased at all concentrations ( figure 2 ) . however , the weight increments were only significant in the 1000 mg / kg for 14 days as well as the 5000 mg / kg for 21 days . treatment of rats with agnps caused elevation ( p < 0.05 ) in total protein levels in rat liver ( figure 3 ) . in contrast , the levels of total protein were reduced in rat serum , kidney , and heart for groups treated with agnps . oral exposure of rats to agnps at various concentrations inconsistently altered the serum and tissue levels of alp relative to the control ( figure 4 ) . in the serum , level of alp increased with increasing length of treatment administration . in contrast , nonsignificant reductions were recorded for tissue levels of alp caused by agnps treatments . for all the agnps treatment groups , the rat serum and tissue levels of ast were inconsistently affected relative to the control ( figure 5 ) . conversely , there was significant ( p < 0.001 ) reduction in the levels of alt in rat serum which was dose and duration dependent ( figure 6 ) . on the other hand , the rat kidney and heart levels of alt were not significantly altered by agnps treatment relative to control . humans can be exposed to these nanomaterials via a number of routes with the nanoparticles tending to accumulate in vital organs . according to gatti , nanoparticle deposition in vital organs or tissues the death could have been accidental . however , treatment at 5000 mg / kg agnps caused dullness in rats . the physical observation supports previous report which reported no death and no effect on the percentage weight gain between the control and treatment groups of mice orally given 5,000 mg / kg of colloidal agnps . , enzyme assays play a crucial role in toxicological evaluation . in the past several years , serum aminotransferases analyses have become a standard measure of hepatotoxicity because of the significance of these enzymes . normally , these enzymes are present in the liver and other tissues where they function in energy metabolism involving the transamination of amino acids . however , in cases of cellular damage , the ast and alt could leak out into the general circulation leading to elevated activity [ 2426 ] . in the present study , the treatment of rats with 100 mg / kg agnps decreased ( p < 0.001 ) levels of ast and alt enzyme significantly , whereas there were increases ( p < 0.001 ) in ast levels in serum , kidney , and heart for the 14- and 21-day treatment groups . treatment of rats with 1000 mg / kg and 5000 mg / kg agnps led to a significant decrease in alt levels and a significant increase in ast levels for the 14- and 21-day treatment groups . in contrast , ast levels significantly decreased while alt levels increased in rat serum treated with colloidal agnps for 7 days when compared with those treated for 21 days . this study also caused elevated ( p < 0.05 ) levels of rat serum alp levels . the effect of the oral administration of agnps to rats on alp , ast , and alt did not follow a definite pattern but may suggest partial inactivation of enzyme activity or depression of enzyme synthesis . although the liver has been reported as one of the recurrent target organs and a dominant site of accumulation of nanoparticles , the alterations to the levels of enzymes may represent adaptive mechanisms by the animals trying to offset stress imposed by exposure to the agnps . the dosages at 100 mg / kg produced more significant alterations to the biochemical parameters than did the higher dosages at 1000 and 5000 mg / kg , respectively . however , a previous study has demonstrated that administration of agnps to rats caused significant alterations to the alp levels . furthermore , alterations to the levels of ast , alt , or alp may not be unconnected with the affinity of agnps for thiol groups in protein molecules . moreover , a separate study by srivastava et al . has also demonstrated the potential of agnps to affect the activity of transaminase enzymes . in separate studies , the potential of silver nanoparticles to modulate enzyme activity was attributable to their affinity for thiol groups [ 15 , 16 ] . it is probable that thiol groups in the enzymes made them attractive to the agnps leading to formation of complexes and consequent modulation of enzyme activity . furthermore , the pronounced biochemical alterations noticed with the 100 mg / kg agnps in the present study may not be completely consistent with earlier report in which agnps had an ld50 of > 5000 mg / kg in mice . the administration of agnps in rats did not induce major changes in body weight and feed intake . however , this study is evidence indicating that rat tissue biochemical indices were altered following oral exposure to agnps . future studies are ongoing and target the determination of whether the alterations to the biochemical parameters were signs of agnps toxicity or not .

objective . to evaluate , in three spanish tertiary referral centres , the short - term safety and efficacy of golimumab ( glm ) for treatment of immune - mediated uveitis resistant to previous immunosuppressive therapy . methods . nonrandomized retrospective interventional case series . thirteen patients with different types of uveitis that were resistant to treatment with at least 2 previous immunosuppressors were included in this study . all included patients were treated with glm ( 50 mg every four weeks ) during at least 6 months . clinical evaluation and treatment - related side effects were assessed at least four times in all included patients . results . eight men and 5 women ( 22 affected eyes ) with a median age of 30 years ( range 2038 ) and active immune - mediated uveitides were studied . glm was used in combination with conventional immunosuppressors in 7 patients ( 53.8% ) . glm therapy achieved complete control of inflammation in 12/13 patients ( 92.3% ) after six months of treatment . there was a statistically significant improvement in mean bcva ( 0.60 versus 0.68 , p = 0.009 ) and mean 1 mm central retinal thickness ( 317 versus 261.2 , p = 0.05 ) at the six - month endpoint when compared to basal values . no major systemic adverse effects associated with glm therapy were observed . conclusions . glm is a new and promising therapeutic option for patients with severe and refractory uveitis . background . silver nanoparticles have found wider and increasing biomedical applications due to their broad antimicrobial characteristics . however , toxicity of nanoparticles is a subject of continued controversy , thus necessitating further studies in this direction . objectives . this study investigated the biochemical effects of silver nanoparticles in wistar rats . materials and methods . forty male rats were randomly distributed into eight experimental groups of five . group a served as the control and received distilled water . groups b to h were orally exposed to varying concentrations of silver nanoparticles ( agnps ) at 100 , 1000 , and 5000 mg / kg daily for 7 , 14 , and 21 days alternately . following cessation of treatments , rats were sacrificed and the blood and other vital organs were collected and prepared as specimens for biochemical analysis . results . administration of agnps to rats did not produce significant loss in feed intake and body weight . however , rat exposure to agnps caused significant alterations to levels of serum and tissue ast , alt , and alp . at the 100 mg / kg agnps exposure , rat serum and tissue ast and alt levels were significantly decreased ( p < 0.05 ) . in contrast , agnps administration elevated ( p < 0.05 ) alp levels in rat serum and tissues . conclusion . we show evidence that agnps administration to wistar rats altered some biochemical parameters .

polycystic ovarian syndrome ( pcos ) is a heterogeneous group of conditions that include anovulation , hyperandrogenism , and polycystic ovaries . the nih - national institute of child health and human development conference of pcos originally recommended that the major criteria for pcos should include hyperandrogenism and/or hyperandrogenemia , oligoovulation , ultrasound pattern of polycystic ovaries and the exclusion of other known disorders . a pcos diagnosis is considered a hyperandrogenic disorder of exclusion with an ovarian etiology . in 2003 , the rotterdam consensus expanded the diagnostic criteria to include at least two of the following three features : clinical and/or biochemical hyperandrogenism , oligoanovulation , and polycystic ovaries , excluding any other endocrinopathies . in 2006 , the androgen excess - pcos society recommended that pcos be defined by clinical and/or biochemical hyperandrogenism , with either oligoanovulation and/or polycystic ovaries , excluding related disorders . the circulating total and free testosterone and dehydroepiandrosterone sulfate ( dheas ) levels are elevated in 5075% of women with pcos , if high quality assays are used . androgen excess is considered by some investigators to be the key feature of pcos ; however , only 8085% of women with clinical hyperandrogenism have pcos [ 4 , 5 ] . despite this evidence hyperandrogenism remains an important part of pcos and is included as major set of symptoms in both nih and rotterdam criteria . according to the nih criteria pcos affects 610% of women of the child bearing age [ 610 ] . as per the broader rotterdam criteria even more individuals can be classified as having pcos . therefore pcos becomes one of the most common human disorders and the single most common endocrinopathy in women of reproductive age . according to a common view pcos is a multifactorial and polygenic in nature [ 1 , 11 , 12 ] . up to now but the question still remains unanswered as to what is primary or secondary : metabolic syndrome or hyperandrogenism . there is an opinion and data suggests that testosterone was significantly related to metabolic syndrome and its components in obese adolescent girls [ 13 , 14 ] . other data reveals that in obese adolescent girls pcos does not add additional risk of further complications of metabolic syndrome [ 15 , 16 ] . pcos adolescent girls have been found to have increased visceral abdominal fat [ 16 , 17 ] , but there was no correlation between visceral abdominal tissue and total testosterone or free androgen index [ 15 , 16 ] . there are case studies that show that low hdl cholesterol is the criterion which best explains the high prevalence of the metabolic syndrome in pcos subjects which , in turn , is influenced by hyperinsulinemia , rather than by hyperandrogenemia . in both animal and human models the associations between serum testosterone and insulin resistance or metabolic syndrome / type 2 diabetes / pcos risk in women were demonstrated , but their cross - sectional nature did not allow conclusions about causality . as such the aim of our study was to find out whether elevated testosterone level impacts the presentation of metabolic syndrome in obese adolescent girls . we enrolled 107 adolescent girls from the pediatric endocrinology clinics and adolescent clinic at suny downstate medical center . pcos group was selected based on nih criteria including irregular menses and clinical and biochemical signs of hyperandrogenism . congenital adrenal hyperplasia , cushing 's syndrome , severe insulin resistance , and androgen - secreting tumors were excluded in all cases . also excluded from this study were patients under treatment with any lipid - lowering drugs , insulin sensitizers ( e.g. , metformin ) , or inositol supplements . the control group was selected from the adolescent clinic patients seen for routine health examinations . high testosterone ( ht ) : patients that met the nih and rotterdam criteria for pcos and had total testosterone > 90 ng / dl ; intermediate testosterone ( it ) : patients that met the nih and rotterdam criteria for pcos and had total testosterone in range 5190 ng / dl ; obese and normal testosterone ( ont ) : patients that did not fulfill the nih and rotterdam criteria for pcos and had bmi > 95th percentile , total testosterone < 50 ng / dl ; controls ( c ) patients that did not fulfill the nih and rotterdam criteria for pcos and had bmi < 95th percentile , total testosterone < 50 and regular periods , and did not meet any of the exclusion criteria . the group of 54 girls who had pcos according to nih criteria including irregular period or amenorrhea and increased level of testosterone was subdivided into 2 subgroups with ( a ) 23 patients with high level of testosterone ( t ) and ( b ) 31 patients with intermediate level of testosterone ( t ) . as a control we assigned 15 patients who had normal bmi and normal level of testosterone . data was obtained for blood pressure , bmi , testosterone , lh , fsh , prolactin , fasting lipid profile , and glucose . bmi was calculated as weight ( kg)/(height ( cm ) ) and bmi above 95th percentile according to the cdc , bmi charts was defined as obese . testosterone was determined by chemiluminescent immunoassay methodology ; high testosterone was defined as testosterone above 90 ng / dl and high intermediate as between 51 and 90 ng / dl . two - sample t - test assuming unequal variances analysis was performed to compare the means of the studied parameter between the 4 groups . multivariate logistic regression analysis was done for testosterone levels and lh , fsh , prolactin , lipid profile , fasting glucose , sbp , dbp , and bmi . for the statistical analysis we used the spss 19.0 software . there was no statistical difference between the high testosterone group ht and the other groups it and ont in sbp , dbp , fasting blood glucose , lipid panel , lh , fsh , and prolactin . furthermore our analyses did not yield any statistically significant difference between the intermediate testosterone group it and ont in any of the studied parameters ( table 1 ) . the control group had lower sbp and bmi comparing with the other groups but no statistical difference was found in dbp , fasting blood glucose , lipid panel , lh , fsh , or prolactin ( figure 1 ) . the regression analysis did not yield any correlation between the testosterone levels and any of the other parameters . the regressions were run both for the entire population studied and for each separate subgroup . bmi had statistically significant correlations with sbp , dbp , cholesterol profile , and lh but no correlation was seen for triglycerides , fsh , and prolactin ( table 2 ) . upon review of the literature , several studies suggest that pcos and high testosterone is an additional risk and is significantly related to metabolic syndrome and its sequelae in obese adolescent girls [ 2125 ] . pcos in adolescent patients has been found to have increased visceral abdominal fat , but there was no correlation between visceral abdominal tissue and total testosterone or free androgen index . an elevated risk profile for cardiovascular morbidity was demonstrated in pcos patients , mainly attributed to increases in type 2 diabetes , dyslipidemia , and hypertension [ 18 , 26 , 27 ] which might constitute a cofounder when studying the correlation between hyperandrogenism and the risk of metabolic syndrome in pcos . however , contrary with such studies [ 28 , 29 ] , we found that there were no significant differences in the relationship between the degree of hyperandrogenism and the metabolic parameters associated with metabolic syndrome . the level or degree of hyperandrogenism did not alter the metabolic parameters in our study group . while it is known that pcos is significantly more prevalent among obese patients and altered metabolic parameters are directly associated to degrees of bmi , there is still no consensus as to the question of what is primary or secondary metabolic syndrome or hyperandrogenism . pcos is polygenic condition and may be influenced by environmental factors like diet and lifestyle . in first - degree female relatives of pcos patients , it has been documented to be an increase in adrenal androgens and insulin resistance in these girls thus suggesting a genetic component . however , to date , there is no clearly defined genetic etiology that is associated with pcos and it is not clear whether hyperandrogenism is involved in the pathogenesis of this disorder . we may speculate that increased rate of metabolic syndrome among obese girl with pcos may be confounded with obesity rather than hyperandrogenism . another reason for lack of correlation between pcos and metabolic syndrome in adolescent girls may be that patients should be exposed to androgens longer to develop metabolic abnormalities in the adult age . it is important for further studies to investigate the effects of prolonged exposure to high circulating androgens mainly in lean pcos patients and the progression of this phenotype to metabolic syndrome . it is our belief that there may be an associated pathophysiological link with obesity , particularly visceral adiposity , and related adipose tissue factors , coagulation abnormalities , or insulin resistance [ 16 , 19 , 32 ] that may independently increase cardiovascular risk rather than a direct association of hyperandrogenism with metabolic syndrome morbidities . there can be limitations arising from the sample collection method or the chemiluminescent immunoassay method used to determine testosterone levels . most common cause of lower gastrointestinal hemorrhage ( lgib ) in elderly patient is diverticular disease and mortality rate is very low 24% . a massive lgib from primary ilio - rectal fistula is rare and difficult to diagnosis but more common from secondary after previous vascular surgery a variety of treatment options are available , such as in situ graft , closure of defect alone , endovascular stent graft , extra - anatomical bypass and embolic coiling . exploration using laparotomy for ligation internal iliac aneurysm and rectal resection is a simple operative procedure which can be applied when emergency option is needed to save a life . we describe successful emergency surgery in primary ilio - rectal fistula when patient presents a massive lgib and hypovolemic shock . an 88-year - old man , his underlying diseases are hypertension , dyslipidemia , moderate aortic stenosis and mitral valve stenosis , chronic kidney disease and prostate cancer . at emergency department , physical exam showed hypotension blood pressure 70/50 mmhg and per rectal exam showed blood clot with enlarged prostate . after resuscitation until stable vital sign , he was sent for colonoscopy but can not be evaluated due to a lot of blood clots in the rectum . he was admitted for observation and we planned to repeat colonoscopy again following day because he was stable and bleeding was stopped . following day after admitting , he had massive lower gastrointestinal bleeding again fresh blood ~1000 ml from his anus . but the patient had a problem vascular access site and then send to computed tomography angiography ( cta ) for evaluate access site . we found 3 cm aneurysm of right internal iliac artery with partial thrombus and attached to rectosigmoid colon with leakage of contrast into sigmoid lumen ( figs 12 ) . he was sent to operating room during transferring we gave blood transfusion and then emergency explore laparotomy was performed , the operation performed by partial aneurysmectomy , ligated right internal iliac , wedge resection rectum wall and temporary abdominal closure with swab packing due to bowel distension and abdominal pressure was high . he was sent to operating room for change swab packing on day 3 and permanent abdominal closure on day 5 . during postoperative period primary aorto / ilio - enteric fistula ( paef ) is very rare , but secondary fistula is more common . sir astley cooper was the first to describe a paef as a condition caused by spontaneous erosion of the aorta into the gastrointestinal tract . etiology of paef are several causes , most causes are due to aneurysm , infection , tumor , radiotherapy and foreign body ingestion [ 59 ] . the most common site of fistula located in duodenum 54% , esophagus 28% , small bowel and large bowel 15% . the classical triad of symptoms associated with a fistula is gastrointestinal hemorrhage , abdominal pain and a pulsating abdominal mass . most common sign and symptoms of fistula is gastrointestinal bleeding , but only 11% presented with classical triad . the diagnosis of paef is difficult to make . because most of patients were send to endoscope due to gastrointestinal bleeding symptoms . several diagnosis investigations were used when patients are clinically stable such as cta , angiography , colonoscopy , and red blood cell scan . all of these do not constitute specific investigation . however , ct scan can diagnose specifically aorto / ilio - enteric fistula ( for example , air within aortic wall or contrast fill in bowel lumen ) that detection rate 61% when failure diagnosis from endoscopy . such as , in situ graft , closure of defect alone , endovascular stent graft , extra - anatomical bypass and embolic coiling . optional treatment depends on surgeon s experience , hospital facilities and the type of paef . this case was send to the operating room after diagnosis right internal ilio - rectal fistula for emergency exploration with laparotomy because the patient s active bleeding and vital signs were not stable preventing adequate preparation time for intervention or stent graft in hospital . we decided to ligate right internal iliac artery , wedge resection rectum with suture repair because this is a simple operation and can save patient s life . other options could be done by endovascular therapy or embolic coiling if we had time to prepare and the patient was already stabilized in this hospital . in conclusion , paef is very rare . the patient who has massive lower gastrointestinal bleeding with failure of endoscope may need to consider imaging to rule out paef . there are several treatment options for paef but no conclusive specific treatment it depends on patient s situation and physician s experience . although endovascular or embolization is minimally invasive surgery and good for the patient who is a high operative risk but an emergency simple operation is an effective procedure not only saves patient s life but also can be done by a general surgeon ( fig . figure 1:axial view iliac aneurysm with partially thrombose contact rectosigmoid colon and contrast leak.figure 2:coronal view iliac aneurysm.figure 3:cta after 1 year .

background . underlying insulin resistance and/or obesity has clearly been implicated in the development of metabolic syndrome in adolescents and young adults with polycystic ovarian syndrome ( pcos ) . it is not clear however what role hyperandrogenism has on the development of metabolic syndrome or its role on those metabolic parameters associated with metabolic syndrome . methods . we studied 107 adolescent girls ; 54 had pcos according to nih criteria . data was obtained for systolic and diastolic blood pressure ( sbp and dbp ) , body mass index ( bmi ) , total testosterone ( t ) , luteinizing hormone ( lh ) , follicle - stimulating hormone ( fsh ) , prolactin , fasting lipid profile , and glucose . the pcos group was divided initially into subgroups according to bmi ( kg / m2 ) , then based on t ( ng / dl ) levels as follows : high testosterone pcos ( ht ) , intermediate testosterone pcos ( it ) , obese and normal testosterone ( ont ) , and lean and normal t ( control , c ) . t - test analysis was performed in between all the groups . results . there was no statistical difference between ht and it , ht and ont , or it and ont in sbp , dbp , fasting blood glucose , lipid panel , lh , fsh , and prolactin levels . the control group had lower sbp and bmi comparing with ont , it , and ht groups . there were no statistical differences found in dbp , fasting blood glucose , lipid panel , lh , fsh , or prolactin . conclusion . metabolic profile in adolescent girls with pcos is not affected by either the presence of hyperandrogenism or the degree of hyperandrogenism . abstractmassive lower gastrointestinal from primary isolated ilio - rectal fistula is a rare condition and difficult to diagnosis and emergency surgery for this situation has a high mortality rate . this report describes a successful operation in an 88-year - old man at present with massive lower gastrointestinal hemorrhage from ilio - rectal fistula and hypovolemic shock . underlying diseases are prostate cancer , hypertension , dyslipidemia , aortic stenosis , mitral valve stenosis and chronic renal disease . operative treatment is to explore laparotomy with internal iliac artery ligation and rectal resection . during postoperative period patient developed acute cholecystitis and treated by cholecystectomy 1 month after operation the patient went home without morbidity and 1-year follow - up he had not any complications .

diabetes , obesity , and cardiovascular diseases are interrelated , with diet being one factor that links them all together . specifically , the current obesity epidemic has been suggested to be a result of increased carbohydrate intake , while dietary fat has frequently been touted as the primary dietary culprit in the cause of many deleterious metabolic conditions . as a nation , we increased carbohydrate consumption during the latter years of the past century as the obesity epidemic began to surge . while it is laudable to try to change the behavior of individuals so they choose other foods , it might be more effective in the short term to address this nutritional issue by providing bioactive compounds that behave like traditional starch yet elicit more favorable metabolic outcomes ( acutely and chronically ) . in other words , let people continue to choose some of the foods they prefer , but make those foods healthier by incorporating bioactive ingredients . to that end , incorporating resistant starches into foods by substituting them for the typical starch has been shown to acutely decrease postprandial glucose and insulin . there are five types of resistant starch , with rs types 2 , 3 , and 4 tending to be studied more frequently . gram for gram , some resistant starches have been reported to elicit minimal glucose and insulin excursions compared with similar amounts of dextrose and are easily incorporated into regular food items with minimal aversion by consumers . given the number of resistant starches commercially available , there is a paucity of evidence from human trials illustrating the effects various types of resistant starches have on glucose metabolism . likewise , some consumers are looking to food to assist them with regulating food intake and/or regulating blood glucose . there is evidence that resistant starch might be able to achieve this by affecting satiety and subsequent food intake , and attenuating postprandial glycemia and insulinemia [ 2 , 5 ] . to complicate matters , behall et al . reported that it is imperative that individual starches and fibers be tested for efficacy given the unique structure and function qualities of starches that are quickly becoming available . for example , haub et al . observed that specific forms of rs2 and rs4 elicit different glycemic responses when compared at the same dose . while cross - linked rs4 has been shown to elicit significant reductions in glycemia and insulinemia , newer versions are being formulated and might provide even greater and/or different health impacts . one new version of rs4 is commercially available but has not been clinically tested to determine its efficacy to assist with regulating blood glucose and minimizing hyperglycemia . therefore , the purpose of this study was to determine the glycemic and satiety effects of novel forms of rs4 , derived from potato starch . the hypotheses were that consuming the rs4 treatments would not affect glycemia compared with a dextrose - only control solution ; and would increase ratings of fullness when added to dextrose control solution . the institutional review board of kansas state university approved the study , and written informed consent was obtained from all volunteers prior to the study . inclusion criteria were no diagnosis of acute or chronic metabolic and cardiovascular diseases , free of gastrointestinal disorders , body mass index of 2030 kg / m , and nonsmokers . volunteers completed five trials via a single - blind randomized crossover design ( we did not alter the water to match the taste / color of the dextrose solution ) . however , neither the subject nor the technician knew which rs treatment they were receiving . during each visit , volunteers consumed one of the following : dextrose solution of 50 g oral glucose tolerance beverage ( dex ) , 38 g of pf mixed into dex ( pf+ ) , 38 g of pr mixed into dex ( pr+ ) , 38 g of pf mixed in to water ( pf ) , and 38 g of pr mixed into water ( pr ) . the doses of rs were chosen to provide 30 g of resistant starch as 80% of each resistant starch was determined to contain rs and dietary fiber ( table 1 ) . the resistant starches were selected as they are novel , were uniquely produced using proprietary techniques , and had not been clinically tested for efficacy regarding their effects on glycemia as resistant starches do not necessarily elicit the same glycemic response . the treatments were randomly administered in crossover fashion using a latin square design . after providing informed consent , as approved by the university research compliance office at kansas state university , volunteers were enrolled into the study . for testing the fasting capillary blood samples were drawn by finger stick prior to drinking the assigned treatment for that day . the remaining blood samples were collected at 30 , 45 , 60 , 90 , and 120 min after ingesting the treatment . blood samples were analyzed in duplicate for glucose concentration using a ysi 2300 stat ( yellow springs , oh ) . after blood was collected , the participants were provided a visual analog scale ( satiety labeled intensity magnitude ( slim ) scale ) to provide a rating of fullness ( rof ) as used by others . volunteers were not shown previous surveys before the next treatment . at the conclusion of the study , one technician ( blind to treatments ) recorded the individual rof responses . the incremental area under the curve ( iauc ) was calculated for glucose using the trapezoid model ( prism v5 , graphpad ) . a repeated measures analysis of variance was used to determine the presence of significant main and interaction effects , and bonferroni post - hoc assessments were performed to determine between treatment effects and significance set at p = 0.05 ( prism v5 , graphpad ) . there was a significant ( p < 0.05 ) difference between the two water trials and three dextrose trials , with the water + rs trials eliciting a significantly decreased glycemic response compared with the three dextrose trials ( figure 1 ) . there were no treatment differences ( p > 0.05 ) within the three dex treatments ( dex , pr+ , and pf+ ) or within the two water treatments ( pr and pf ) . there was a significant main effect for time in pr+ , pf+ , and dex , while there were no time effects following the ingestion of 38 g of each resistant starch mixed with water . the pr+ and pf+ treatments with 38 g of potato - derived modified starches added to dex did not increase the glycemic response compared with dex alone . this study illustrates that two novel potato - derived resistant starches did not elicit a glycemic response when added to 296 ml of water . likewise , there was no treatment effect of each rs4 added to dex when compared to dex alone . the glycemic results of the present study are similar to data from our previous assessment of a wheat - derived rs4 in that the excursion from baseline was minimal when pf and pr were ingested when added to water . the acute glycemic effect of resistant starch , regardless of type , has been studied extensively and has been shown to decrease glucose and insulin when ingested as part of a food [ 2 , 6 , 9 ] , but few studies to date have concomitantly investigated the effects on satiety . a study by raben et al . , who also used potato starch , had volunteers ingest meals containing raw or pregelatinized potato starch on postprandial glucose and insulin concentrations and satiety . they observed that the replacement of raw potato starch ( increased rs content ) for pregelatinized starch elicited decreased glucose and insulin levels . contrary to the present observations , they reported that the resistant starch meal elicited a significant ( p < 0.05 ) decrease in fullness and satisfaction . the difference between their observations and those of the present study are likely due to the methods of incorporating the resistant starch into the treatments that were ingested . raben et al . replaced digestible starch with resistant starch , while we added the starch to the digestible carbohydrate . thus , the treatments used by raben et al . did not use treatments that had equal amounts of readily digestible starch . since the resistant starch treatment had 50% less energy from readily digestible starch , their decreased postprandial glucose and insulin responses are expected and are in agreement with the glucose differences observed in the present study between the pf and pr treatments when compared with dex , pf+ , and pr+ treatments , as the pf and pr treatments did not contain 50 g of dextrose . the difference in satiety results reported between studies may , also , be due to the energy differences of the meals reported by raben et al . , as their rs treatment was reported to contain 367 kj , while the pregelatinized starch meal contained 917 kj . however , a study by leeman et al . supports the satiety responses observed in the present study as they reported no difference in ratings of satiety for potato foods ( french fries and boiled potatoes ) with differing levels of energy content , indicating that factors other than energy content , such as energy density , may need to be taken into account when assessing satiety and subsequent energy intake . the present data , also , support the notion that postprandial glycemia and satiety responses may not always coincide . even though subjects were consuming more energy with each dextrose treatment ( pf+ , pr+ , and dex ) , the reported satiety responses were not different from those observed with pf and pr. this observation has been previously reported . an explanation for the lack of an rof effect might be that the beverages did not reach a portion threshold to elicit a satiety response . the limitations of this study are as follows : the starches were not incorporated into a food ( bread , muffin , etc . ) , rof could have been influenced by volume of intake as the pf+ and pr+ treatments ( 88 g of carbohydrate ) had a greater volume than the dex trial ( 50 g of carbohydrate ) , the sample size was powered for glycemia and not fullness , only one outcome of satiety was assessed . even with shortcomings , this study is significant as it illustrates that glycemia did not influence rof indicating that beverage - induced increases in blood glucose do not appear to impact rof . future studies need to measure intake overtime and take more measures of satiety to better understand how results from acute studies translate to habitual eating . in conclusion , this study presents novel data regarding the combined physical ( glucose ) and psychological ( satiety ) effects of a potato - derived resistant starch . these data indicate that 38 g of pf and pr added to water do not affect capillary glucose over two hours . likewise , when 38 g of pf and pr were added on top of 50 g of dextrose ( 88 g of carbohydrate ) , those resistant starches did not affect glucose or satiety responses when compared with the dex ( 50 g of carbohydrate ) treatment . since the addition of these starches did not affect blood glucose , these rs products might be useful substitutions for standard rapidly digestible starches to decrease postprandial glucose , and likely insulin , to better control blood glucose when incorporated into a food product . however , those foods would need to be clinically tested to insure the effects translate . enamel subsurface lesions need to be diagnosed as early as possible for the best possible remineralization of enamel and restoration as well as a function of the tooth . there is evidence based on the remineralization potential of agents such as fluoride , casein phosphopeptide amorphous calcium phosphate ( cpp - acp ) , casein phosphopeptide amorphous calcium phosphate with fluoride ( cpp - acpf ) . it has been observed that cpp - acp remineralized initial enamel lesions and has shown increased remineralization potential when used along with fluoridated toothpaste . apf gel forms more fluoride in enamel than neutral gel and it is more efficient in reducing enamel demineralization of enamel blocks submitted to cariogenic challenge than the neutral one . cpp - acpf is proven to produce significantly greater mean percentage of remineralization in comparison with cpp - acp . mineral deposition throughout the lesion has been proven in relation to use of both cpp - acp and cpp - acpf . it has been observed that gums comprising of cpp - acp nano complexes have a marked remineralization potential . the cpp - acp has also been shown to remineralize enamel subsurface lesions in situ when delivered through oral care products . the probable anticariogenic mechanism of cpp - acp is its ability to localize acp at the tooth surface , which brings about buffering of calcium and phosphate free ion activities , thereby helping to maintain a state of super saturation with respect to tooth enamel negating demineralization and enhancing remineralization . cpp - acpf due to its added fluoride content has shown improved ability to remineralize initial caries . in incipient enamel caries low concentrations of fluoride are able to penetrate deeper into the body of the lesion and bring about remineralization , hence sodium fluoride ( naf ) ( 200 ppm/0.044% ) has been opted for use in this study . the study aimed at quantitatively evaluating the enamel remineralizing potential of cpp - acp , cpp - acpf and naf using surface microhardness analysis ( vickers hardness test ) . the teeth were cleansed of visible blood and gross debris and were maintained in a hydrated state during storage . extracted teeth were placed in sodium hypochlorite solution diluted with saline in a ratio of 1:10 in the container with a secure lid to prevent leaking and labeled with the biohazard symbol . teeth that do not contain amalgam restorations were preferred because they can be safely autoclaved . however , extracted teeth containing amalgam restorations , were immersed in 10% formalin solution for 2 weeks . enamel samples ( 2 mm thickness ) were prepared from the buccal and lingual surfaces of the teeth selected , using a double faced diamond disc mounted on a contra - angle hand piece . following sample preparation windows were created ( dimension of 5 mm 5 mm ) using adhesive tape and the sample was made completely resistant to acid attack by coating nail varnish . the adhesive tape was removed from the enamel using a sharp tipped instrument exhibiting a rectangular area on the enamel surface . a total of 50 enamel slabs were randomly divided into five groups of 10 samples in each based on the type of remineralizing agent to be used [ table 1 ] . the division of groups each of the enamel samples were then immersed in 40 ml of demineralizing solution ( acetate 0.1 mol / l , calcium 0.1 mol / l , phosphate 0.1 mol / l , fluoride 0.1 mg / l , ph 5.0 ) for a period of 4 days at a constant temperature of 37c , in an incubator to induce artificial caries formation , simulating an active area of demineralization . slurry of cpp - acp and cpp - acp along with fluoride were prepared by agitating the above mentioned preparations in deionized water in the ratio of 1:3 . ph cycling model was adopted to simulate the dynamic process of demineralization and remineralization that occurs in the oral cavity . each of the enamel samples were treated with the respective remineralizing agents for a period of 2 min , following which the samples were individually immersed in 20 ml of demineralizing solution ( calcium 2.0 mmol / l , phosphate 2.0 mmol / l , acetic acid 75.0 mmol / l , ph 4.4 ) for a period of 3 h. this was followed - up with treatment of the samples again with slurries of the respective remineralizing agents for 2 min . all the enamel samples were individually immersed in 30 ml of remineralizing solution ( 1.5 mm calcium , 0.9 mm phosphate , 0.15 m kcl in 0.1 m tris buffer , ph 7 ) for a period of 17 h. the remineralizing solution was replaced every 48 h and the demineralizing agent replaced every 5 days . the ph cycling was carried out for a period of 28 days . after the completion of the process of ph cycling , all the groups of enamel samples were assessed for surface microhardness using vickers hardness test ( micro vickers hardness tester , matsuzawa co. , ltd , toshima , japan ) . the surface microhardness of the specimens was determined using digital microhardness tester ( matsuzawa co. , ltd . model - mmt x7 , japan ) with a vickers elongated diamond pyramid indenter and a 40 objective lens . a load of 100 g was applied to the surface for 10 s. five indentations were placed on the surface and the average value was considered . the diagonal length of the indentation was measured by built in scaled microscope and vickers values were converted to microhardness values . multiple comparisons between groups were performed by posthoc tukey test [ tables 2 and 3 ] . the teeth were cleansed of visible blood and gross debris and were maintained in a hydrated state during storage . extracted teeth were placed in sodium hypochlorite solution diluted with saline in a ratio of 1:10 in the container with a secure lid to prevent leaking and labeled with the biohazard symbol . . however , extracted teeth containing amalgam restorations , were immersed in 10% formalin solution for 2 weeks . enamel samples ( 2 mm thickness ) were prepared from the buccal and lingual surfaces of the teeth selected , using a double faced diamond disc mounted on a contra - angle hand piece . following sample preparation windows were created ( dimension of 5 mm 5 mm ) using adhesive tape and the sample was made completely resistant to acid attack by coating nail varnish . the adhesive tape was removed from the enamel using a sharp tipped instrument exhibiting a rectangular area on the enamel surface . a total of 50 enamel slabs were randomly divided into five groups of 10 samples in each based on the type of remineralizing agent to be used [ table 1 ] . each of the enamel samples were then immersed in 40 ml of demineralizing solution ( acetate 0.1 mol / l , calcium 0.1 mol / l , phosphate 0.1 mol / l , fluoride 0.1 mg / l , ph 5.0 ) for a period of 4 days at a constant temperature of 37c , in an incubator to induce artificial caries formation , simulating an active area of demineralization . slurry of cpp - acp and cpp - acp along with fluoride were prepared by agitating the above mentioned preparations in deionized water in the ratio of 1:3 . ph cycling model was adopted to simulate the dynamic process of demineralization and remineralization that occurs in the oral cavity . each of the enamel samples were treated with the respective remineralizing agents for a period of 2 min , following which the samples were individually immersed in 20 ml of demineralizing solution ( calcium 2.0 mmol / l , phosphate 2.0 mmol / l , acetic acid 75.0 mmol / l , ph 4.4 ) for a period of 3 h. this was followed - up with treatment of the samples again with slurries of the respective remineralizing agents for 2 min . all the enamel samples were individually immersed in 30 ml of remineralizing solution ( 1.5 mm calcium , 0.9 mm phosphate , 0.15 m kcl in 0.1 m tris buffer , ph 7 ) for a period of 17 h. the remineralizing solution was replaced every 48 h and the demineralizing agent replaced every 5 days . the ph cycling was carried out for a period of 28 days . after the completion of the process of ph cycling , all the groups of enamel samples were assessed for surface microhardness using vickers hardness test ( micro vickers hardness tester , matsuzawa co. , ltd , toshima , japan ) . the surface microhardness of the specimens was determined using digital microhardness tester ( matsuzawa co. , ltd . model - mmt x7 , japan ) with a vickers elongated diamond pyramid indenter and a 40 objective lens . a load of 100 g was applied to the surface for 10 s. five indentations were placed on the surface and the average value was considered . the diagonal length of the indentation was measured by built in scaled microscope and vickers values were converted to microhardness values . multiple comparisons between groups were performed by posthoc tukey test [ tables 2 and 3 ] . in spite of the enamel remineralizing potential of saliva , by itself it fails to initiate the process of increasing the levels of calcium and phosphate . calcium and phosphate ions must first penetrate the surface layer of enamel , to bring about deposition of minerals through the body of the lesion , which confirms the reason for the cpp supported metastable calcium phosphate solutions of being such efficient remineralizing solutions . majority of them are composed of calcium and phosphate with acetic acid or lactate . the main variation lies in concentrations of each component , which influences the final ph and the sample exposure time . the ph employed varies from 3.5 to 5 and the time differs from 2 h to 21 days . in this study demineralization various studies have used different methods to assess the process of enamel remineralization . the commonly used microhardness tests for evaluating enamel remineralization are vickers microhardness test and knoop microhardness test . as this study put the focus on evaluation of surface microhardness of enamel , vickers surface microhardness test was used . the values of surface microhardness indicate that remineralization of enamel is more in samples of group iii ( cpp - acpf ) followed by group iv ( naf ) and group ii ( cpp - acp ) . this may be because of the presence of fluoride in cpp - acp makes it more capable to remineralize the enamel . however , group iv ( f ) showed a marginal increase in remineralization compared with group ii ( cpp - acp ) . one - way anova shows that there is a significant difference between all the five groups . however , multiple group posthoc tukey test showed a significant difference only between groups v with the other groups . no other group showed significance when they were inter - compared . adopting the usage of low fluoride concentration as present in cpp - acpf ( 0.2% ) , there is a complex localization of free calcium phosphate and fluoride ion activities , which aid in maintaining a state of super saturation by suppressing demineralization . even though fluoride is present in tooth pastes manufactured in india and china , research has also proven that apf forms more fluoride and is more efficient in reducing enamel demineralization when compared with neutral gel . sugar free chewing gums containing cpp - acp and citric acid result in higher remineralisation when compared with chewing gums without cpp - acp and citric acid and gums with citric acid alone . research suggest that fluoride dentifrices in combination with daily applications of acidulated phosphate fluoride ( apf ) topical thrice and four times a day were the only treatments able to increase the surface microhardness of enamel . the use of fluoride and the cpp - acp in recent years have been the best possible method in prevention of enamel caries and also in halting the progress of the existent enamel lesions . cpp - acp containing remineralising paste has shown remineralization effect within the first three month evaluation period . conversely , research also suggest that acp - cpp cream is nt as effective as fluoride in remineralizing early enamel caries at surface level . combination of fluoride and acp - cpp does not provide any additive remineralization potential compared to fluoride alone . however , it 's important to note that compliance of patient in oral hygiene maintenance and in home fluoride use is of utmost importance in prevention of enamel caries . the results of this study showed that cpp - acpf remineralized enamel subsurface lesion better in comparison with naf and cpp - acp . naf shown to be better than cpp - acp in enamel remineralization although the comparison between these two agents was insignificant statistically . with this basis we can confirm that cpp - acpf is an excellent material for remineralization of white spot or initial enamel caries . cpp - acp effectively remineralizes initial enamel caries , but to a lesser extent in comparison to cpp - acpf and naf.the addition of fluoride to cpp - acp shows improved remineralization of initial enamel caries when compared with cpp - acp and naf . cpp - acp effectively remineralizes initial enamel caries , but to a lesser extent in comparison to cpp - acpf and naf . the addition of fluoride to cpp - acp shows improved remineralization of initial enamel caries when compared with cpp - acp and naf .

this study was designed to determine the efficacy of two novel type - four resistant starches ( rs4 ) on postprandial glycemia and ratings of fullness . volunteers ( n = 10 ) completed completed five interventions designed to determine the glycemic and satiety ( fullness ) effects of the starches ( 38 g , ) alone and when added on top of available carbohydrate . the dose of the starches provided 30 g of resistant starch per treatment . the treatments were : commercial resistant starch added to water ( pf ) , noncommercial resistant starch added to water ( pr ) , dextrose solution ( dex , 50 g ) , and dex with penfibe starch ( pf+ ) , and dex with the non - commercial starch added ( pr+ ) . blood glucose was measured in the fasted state and following the randomly assigned treatments at 30 , 45 , 60 , 90 , and 120 minutes post - consumption . a visual analog scale was used to determine fullness at each time point . there were no differences in the glucose incremental areas under the curve ( iauc ) for pf+ and pr+ compared with dex . the pf and pr treatments had decreased ( p < 0.05 ) iaucs for glucose compared with dex , pf+ , and pr+ . there were no treatment differences for rof . the dose ( 38 g ) of starches did not to alter glucose responses when added on top of 50 g of dextrose . aim : the aim of this study is to evaluate the enamel remineralization after treatment with three different remineralizing agents using surface microhardness assessment.materials and methods : this in vitro study involves 50 enamel samples divided into five groups of 10 samples each . the positive control group consisted of intact enamel and a negative control group consisted of demineralized enamel samples . all groups excluding the positive control group were subjected to demineralization following which three of these groups were remineralized using remineralizing agents ( casein phosphopeptide amorphous calcium phosphate [ cpp - acp ] [ gc tooth mousse ] , casein phosphopeptide amorphous calcium phosphate with fluoride [ cpp - acpf ] [ gc tooth mousse plus ] , sodium fluoride [ phos - flur ] ) . the groups treated with remineralizing agents were subjected to ph cycling over a period of 28 days . this was followed with assessment of surface microhardness ( micro vickers hardness tester , matsuzawa co. , ltd , toshima , japan).statistical analysis : one - way analysis of variance test and posthoc tukey test were conducted for multiple group comparison.results:there was an improved enamel remineralization in the group , remineralized using cpp - acpf in comparison with the other groups.conclusion:casein phosphopeptide with fluoride is a promising material for remineralization of enamel subsurface lesions .

pancreatic neuroendocrine tumors ( pnets ) represent a relatively uncommon form of malignancy with an incidence of 0.43 cases per 100,000 in the usa . at diagnosis , nearly 70% of patients have metastatic disease , of which 85% will have liver metastases . management options for pnets metastatic to the liver include surgical , ablative , cytotoxic , and radioisotope approaches . unfortunately , due to the scarcity of these tumors there is a paucity of randomized trials to guide optimal therapy sequencing . the north american neuro - endocrine tumor society and european neuroendocrine tumor society both support the use of radioembolization for progressive or symptomatic liver metastasis [ 3 , 4 ] . to date , yttrium-90 ( y ) therapy has appeared safe ; however , there is no randomized controlled trial assessing toxicities . we present the case of a woman undergoing y therapy for metastatic pnet to the liver who developed liver enzyme elevation and subsequent cirrhosis following treatment . there are only 3 other reported de novo cases of cirrhosis following y administration , with only 1 demonstrating confirmatory pathology [ 6 , 7 , 8 ] . ultrasonography and computed tomography ( ct ) of the abdomen revealed a 9.5 8.6 10.5 cm , heterogeneous , hypervascular mass adjacent to the spleen and abutting the stomach wall and tail of the pancreas . fine - needle aspiration guided by endoscopic ultrasound revealed cytologic evidence of a neuroendocrine tumor . the patient proceeded to a distal pancreatectomy , splenectomy , wedge resection of the stomach , and partial resection of the left adrenal gland . pathology demonstrated a 13-cm , well - differentiated neuroendocrine tumor of the pancreas with perineural invasion , but no vascular invasion and negative margins . it was found to be adherent to both the spleen and the stomach , but did not invade either . it had a mitotic rate of 2 mitoses / high - power field and a ki-67 index of < 2% . two months postoperatively , the patient was found to have 4 subcentimeter hypervascular lesions in the liver which were in octreotide scan negative . over the following 9 months , the patient developed 8 new lesions , while the original lesions increased to a maximum size of 1.2 cm . therapy with octreotide lar 20 mg intramuscularly once monthly was initiated but discontinued after 9 months due to progressive hepatic disease . a ct scan of the liver performed 3 months after embolization demonstrated a mixed tumor response with the overall impression of progressive disease and development of new liver metastasis . the patient was presented with the option of systemic therapy with everolimus , shown in phase iii trials to improve progression - free survival in patients with well - differentiated pnets . due to the absence of extrahepatic metastasis , liver - directed therapy with y embolization was also offered , which the patient chose to proceed with . prior to y treatment , there was no radiologic evidence of cirrhosis and the liver enzymes were within normal ranges ( ast 24 u / l [ normal range ( n ) 1038 < 50 u / l ] , alkaline phosphatase 156 u / l [ n 50200 u / l ] , total bilirubin 5 mol / l [ n 018 mol / l ] ) . she had a technetium-99 macroaggregate albumin planning spect ct demonstrating multiple focal regions of increased activity in the left and right lobes of the liver which corresponded to the patient 's known metastases . radioembolization with 3.5 gbq of y theraspheres was administered to the right hepatic artery , delivering a total dose of 140 gy to the right lobe of the liver , and 1.8 gbq of y theraspheres was delivered through the left hepatic artery with a dosing of 140 gy to the left lobe of the liver on the same day . over the following 3 months , the patient developed abdominal pain and nausea and was found to have elevated liver enzymes , in a pattern suggesting damage to the biliary tree ( ast 52 u / l , alt 86 u / l , alkaline phosphatase 405 u / l , ggt 604 u / l , total bilirubin 5 ct scanning of the abdomen demonstrated generalized mottling of the liver , compatible with postembolization edema with a substantial decrease in tumor burden and 2 significant lesions remaining . the patient 's liver enzymes remained elevated over the following 6 months until she developed significant right upper quadrant pain . ct 9 months after y treatment revealed a very inhomogeneous liver texture and a large biloma traversing segments 5 and 8 of the liver , measuring 8.5 6.5 5.5 cm . this was initially noted 10 months after y treatment and was slightly progressive on the 14-month scan ( fig . 1 ) . imaging also demonstrated multiple subcentimeter right hepatic lesions raising concern for metastatic disease , some of which had not previously been seen . due to radiographic evidence of progressive disease , everolimus 10 mg once daily was initiated , and the patient continued on this for 36 months with stabilization of the disease . her liver enzymes remained unchanged over this time , with elevated hepatobiliary enzymes and borderline - elevated hepatocellular enzymes . at 25 months after initiation of everolimus therapy , progressive disease was noted in 2 solitary hepatic lesions and was treated with radiofrequency ablation . due to disease control in the remainder of the hepatic parenchyma , everolimus was resumed after radiofrequency ablation . thirty - five months after initiation of everolimus therapy , radiographic note was made of the advanced cirrhotic appearance of the liver ( fig . 1 ) and the increase in size of a left hepatic lesion . there was no evidence of portal hypertension . due to the radiographic evidence of progressive disease and the cirrhotic appearance of the liver , everolimus therapy was interrupted and a liver biopsy to further characterize the nature of the parenchymal disease was performed . a single 18-gauge core was obtained from the left lobe of the liver in order to avoid the biloma involving the right lobe . the biopsy demonstrated large fibrotic bands in keeping with cirrhosis with mildly active steatohepatitis ( approx . the patient had not been started on any other new medications , did not drink alcohol excessively , performed no high - risk activities for viral hepatitis , and had no family history of liver disease . serology was nonreactive for hbsag , hbcab , and hepatitis c virus , and the patient had immunity to hepatitis b virus . her antinuclear antibody was equivocal , and her anti - smooth muscle antibody and antimitochondrial antibody were negative . given the chronologic association of y administration with liver enzyme elevation , with the exclusion of other causes of cirrhosis , the diagnosis of y - induced cirrhosis was made . she has not suffered any cirrhosis - associated complications and is currently child - turcotte - pugh class a. to date , y - related cirrhosis has only been reported in 3 cases [ 6 , 7 , 8 ] . a prior systematic review assessing outcomes following y radioembolization identified grade 3 and higher adverse events in 012.9% of patients . in their review , the authors were able to identify 1 episode of cirrhosis and 3 episodes of hepatic failure . the 1 cirrhotic patient identified was seen by saxena et al . , who described a patient with cirrhosis and ascites 6 months after treatment with grade 3 elevation of serum alkaline phosphatase and grade 2 elevations of alt and bilirubin . other reported cases of cirrhosis come from case reports by brock et al . and ayav et al . brock et al . described a patient with metastatic breast cancer with bone and liver metastases who received y. the patient had a good response to treatment at 4 weeks , but 3 months later developed ct imaging findings consistent with cirrhosis and portal hypertension . described a 39-year - old man who received y for colon carcinoma metastatic to the liver . four months after administration , the patient was found to have signs of portal hypertension and had a biopsy consistent with cirrhosis . the timeline of initial hepatic changes seen with our patient closely resembles these previous reports , with a subacute onset of irregular echotexture and cirrhosis occurring several months following treatment with y [ 6 , 7 , 8 ] . y microspheres deliver focused radiotherapy to metastases in the liver while sparing viable hepatic tissue , due to the predominant arterial supply of hepatic metastases as compared to the predominantly portal venous blood supply of normal liver parenchyma . an assessment of explanted livers following y treatment showed no fibrosis > 1 cm from tumor borders . the case of our patient adds to 3 previously reported cases that demonstrate cirrhosis as a potential long - term complication of y treatment . an increasingly long median life expectancy is observed with pnets , with a 5-year overall survival of patients with low - grade tumors of 75% . therapies with a significant rate of long - term toxicities should therefore be avoided early in the course of therapy among patients with advanced disease . the notion that y therapy may lead to cirrhosis is supported by radiographic studies assessing volume changes and signs of portal hypertension following y treatment . in a series of 17 patients treated with y , a mean decrease in liver volume of 11.8% and a mean increase in splenic volume of 27.9% were noted , accompanied by increases in portal vein diameter . in another series of 45 patients , liver volumes were shown to decrease in lobes treated with radioembolization by up to 45% at 12 months after treatment . the difficulty with utilizing volume changes and liver texture to define outcomes following y administration lies in differentiating normal radiographic changes following radioembolization from true cirrhosis . in the current case report , imaging was reported as being consistent with prior radioembolization throughout many of the reports , with a progressive worsening noted over time . while differentiating this from cirrhosis is very difficult , the differential for these radiographic changes needs to include cirrhosis . the only other agent that could be chronologically associated with cirrhosis in our patient is everolimus ; however , this was started after radiographic changes had already been seen in the liver . in addition , early preclinical work suggests that inhibition of the mtor pathway with mtor inhibitors may be antifibrotic . there is , however , 1 case report of steatohepatitis following administration of everolimus . while our patient did not have steatosis severe enough to be considered solely responsible for the observed cirrhosis , it is possible that she suffered an initial insult from y administration and had impaired hepatic regeneration subsequently due to everolimus - induced steatosis , thus causing accelerated progression to cirrhosis she developed a large biloma 10 months following y treatment . in a 327-patient retrospective review by atassi et al . in contrast to normal hepatocytes , intrahepatic bile ducts lack a dual blood supply and are fed by intrahepatic arteries . biloma development is thought to develop following peripheral bile duct necrosis , resulting in biliary drainage into surrounding tissue . this case report adds to a growing body of evidence that y treatment may cause serious damage to the biliary tree , and it is clinically important because it demonstrates pathology confirming that a long - term outcome of y administration may be cirrhosis . this long - term risk needs to be considered when sequencing therapy for patients who have a high rate of 5-year survival and who have other systemic and ablative treatment options . comparar las mediciones de la presin intraocular ( pio ) obtenidas con el tonmetro de rebote ( rt ) , el tonmetro de contorno dinmico ( dct ) y el tonmetro de aplanamiento de goldmann ( gat ) en crneas con queratocono , e investigar los efectos del espesor central de la crnea ( cct ) y los radios de curvatura de la crnea ( cr ) en las mediciones de la pio . este estudio transversal se realiz sobre sesenta y tres ojos de un nmero igual de pacientes con queratocono . se midi la pio de cada sujeto siempre en el mismo orden , icare rt - pascal dct - gat , tras un intervalo mnimo de diez minutos entre mediciones . las mediciones de cct y cr se realizaron utilizando una cmara scheimpflug rotatoria , antes de medir la pio en todos los sujetos . se utilizaron los anlisis de coeficiente de correlacin de pearson y la anova de una va de medidas repetidas para realizar la valoracin estadstica . la pio media para todos los ojos estudiados fue de 11,72 2,59 mm hg para gat , 9,34 3,29 mm hg para rt , y 15,42 3,31 mm hg para dct . se produjeron diferencias estadsticamente significativas entre los tres tonmetros ; gat y rt ( p<0,001 ) , gat y dct ( p<0,001 ) , rt y dct ( p<0,001 ) . gat y rt reflejaron una relacin significativamente positiva con cct ( r=0,288 , p=0,025 y r=0,483 , p<0,001 , respectivamente ) . dct no reflej una correlacin significativa con cct ( r=0,115 , p=0,377 ) ni con cr ( r=0,179 , p=0,168 ) . el tonmetro dct sobreestim las mediciones de la pio , y el rt subestim las mismas , con arreglo a las mediciones de gat en las crneas con queratocono . dct ha demostrado ser el tonmetro ms adecuado para utilizar en las mediciones de la pio en los casos de queratocono , ya que no parece depender de cct y cr . proper measurement of intraocular pressure ( iop ) is fundamental in the diagnosis and follow - up of glaucoma because elevated iop is the basic treatable risk factor in the management of glaucoma . the goldmann applanation tonometer ( gat ) is currently the most widely used device in clinical practice , and is accepted as the gold standard method for iop measurement . however , it is known to be affected by changes in corneal thickness , structure , and curvature . keratoconus is a bilateral , progressive , non - inflammatory corneal ectasia that results in irregular astigmatism and reduced vision quality because of corneal thinning and protrusion . hence , the corneal changes associated with keratoconus may potentially lead to difficulties in accurate determination of iop in these patients . previous studies have shown that currently widely used tonometers such as the gat , tend to underestimate iop in keratoconic eyes mainly because of the characteristic reduced corneal thickness . because of the many difficulties in iop estimation in the keratoconic eyes , clinicians are interested in new tonometers that attempt to measure iop independently of corneal thickness and curvature . the icare rebound tonometer ( rt ) is a portable handheld tonometer , which does not require any topical anesthetic . it records iop by detecting the deceleration of a rod probe as it is bounced off the cornea . as the iop increases , the rod probe bounced off the cornea faster rt also minimizes corneal injury and avoids the risk of cross infection through the use of disposable probes . rt has been shown to correlate well with gat and is generally accepted to be dependent on corneal parameters . rt readings are , however , on average , higher than gat readings in previous studies.5 , 6 , 7 the pascal dynamic contour tonometer ( dct ) claims to be relatively unaffected by corneal biomechanical properties . it is a slit - lamp mounted , nonapplanation , contour - matching contact tonometry . the tip of the tonometry has a concave surface and measures iop when the cornea of patient matches the tip of the tonometry . it produces minimal distortion of the cornea and the iop is the result of a direct measurement by a sensor integrated into the center of the tip . the iop and quality of the data ( q1 - 5 ) are reported on a digital display . dct also evaluate ocular pulse amplitude ( opa ) which is the difference between the average systolic and diastolic iops . dct would be theoretically unaffected by neither central corneal thickness ( cct ) nor corneal radius of curvature ( cr).8 , 9 , 10 the purpose of this study was to compare iop measurements obtained by gat , rt , and dct and to assess relationship between iop measurements and cct , cr in keratoconic corneas . this cross - sectional study was conducted at the department of ophthalmology of the eskiehir osmangazi university school of medicine . the study was performed in accordance with the declaration of helsinki principles and the local medical ethics committee approved the study . all study participants underwent a detailed ophthalmologic examination including best - corrected visual acuity , slit - lamp biomicroscopy , and fundoscopy . the diagnosis of keratoconus was made by an experienced corneal specialist ( n.y . ) on the basis of the following diagnostic criteria ( 1 sign or a combination of signs ) : external signs such as munson 's sign ( v - shaped conformation of the lower lid on down gaze ) , biomicroscopic signs such as stromal thinning , conical protrusion , fleischer ring , vogt striae , and enlarged corneal nerves ; and an abnormal retinoscopy reflex . the diagnosis was confirmed topographically with the oculus pentacam system ( tkc topographical keratoconus classification ) . the contact lens wearers stopped using their contact lenses for 1 week before all measurements . patients who had corneas with apical scarring or any kind of surgical treatment for keratoconus were excluded . all iop measurements were taken between the 9 am and 11 am to minimize the potential effect of diurnal variation in iop . iop was measured on each subject in a sitting position and always in the same order , rt - dct - gat , after a minimum interval of 10 min between measurements . icare rt ( ta01i , tiolat oy , helsinki , finland ) is conducted by positioning the tip of the probe in front of the central cornea at a distance of 48 mm before the measurement . the software discards the highest and lowest iop readings automatically and calculates the average iop value from the rest . pascal dct ( smt swiss microtechnology ag , port , switzerland ) is a self - calibrating device mounted on the slit - lamp . it consists of a sensor tip with a 10.5 mm radius of curvature , a concave surface , and a miniaturized pressure sensor integrated into the center of the contact surface . the device displays the iop value accompanied by a quality control ( q1 - 5 ) . measurement was performed under topical anesthesia with one drop of 0.5% topical proparacaine solution ( alcaine , alcon laboratories inc . mean of three qualified iop values ( q result is 1 or 2 ) were considered for statistical analysis in this study . gat ( at900 , haag - streit , koeniz , switzerland ) measurement was performed using a slit - lamp with same topical anesthetic and fluorescein under cobalt blue filtered light . three consecutive readings were obtained moving the probeaway from the cornea after each measurement and a mean iop value was calculated . in all subjects , cct and cr were measured using a rotating scheimpflug camera ( oculus pentacam , wetzlar , germany ) before the iop measurements . the differences between iop readings were compared with the one way repeated measures anova . the relationship between cct , cr , and we use only one eye data per person to avoid problems arising from using dependent measurements ( measurements of both eyes of the same person ) . we choose the eye with the higher mean keratometry reading when the second eye of the same patient has keratoconus . all analyses were performed wih the softwares ibm spss statistics version 21 and medcalc version 12.7.5.0 . the statistical power analysis of our findings was made by using r for windows x64 ( v.3.2.2 ) , a free and powerful statistical analysis software and programming language ( r foundation for statistical computing , vienna / austria , 2016 , https://www.r-project.org ) . as we have had one study group and our findings were mainly based on correlation relationship , we have preferred to use power correlation calculation . the results are presented in the following table : correlation between(n ) case number(r ) correlation valuesignificance levelpower valuegat cct630.1150.3770.5457dct cr630.1790.1680.5171as it can bee understood from the table , calculated power levels of the given correlations are greater than 0.50 threshold cut - off level and this is sufficient for the given number of patients . a total of 63 eyes of 63 patients ( 27 females and 36 males ) with a mean sd age of 33.57 9.41 years ( range , 1961 years ) were enrolled in the study . mean iop measurements obtained by each tonometer , mean cct , and mean cr values are shown in table 1 . comparison of the gat , rt , and dct derived iop measurements are shown in table 2 . dct measurements were significantly higher than gat measurements ( p < 0.001 ) and rt measurements ( p < 0.001 ) . also gat measurements were significantly higher than rt measurements ( p < 0.001 ) . we found significantly positive correlation between gat measurements and cct ( r = 0.288 , p = 0.025 ) . rt measurements were significantly positively correlated both cct ( r = 0.483 , p < 0.001 ) and cr ( r = 0.550 , p < 0.001 ) . however , dct did not correlate with cct ( r = 0.115 , p = 0.377 ) and cr ( r = 0.179 , p = 0.168 ) . rt is the most affected and dct is the least affected tonometer from cct and cr . the gat is the most widely used method of measuring the iop , but corneal parameters , especially corneal thickness , affect the accuracy of this tonometer . so , newer devices such as rt and dct are the new alternative techniques for iop measurements . the initial reports on these new tonometers are promising and the reproducibility or reliability of data is being evaluated . in our study , dct measurements were significantly higher than gat measurements , but rt measurements were significantly lower than gat measurements in keratoconic corneas . rt has recently appeared in clinical practice after being used for some time in animal research . its relatively low cost , portability , lack of need for topical anesthesia , being independent of a slit lamp and ease of use make it ideal for routine clinical practice . previous comparative studies of iop measurements recorded with rt and gat have shown clinical agreement between the two devices , with a slight overestimation of readings with rt when compared with gat.5 , 6 , 7 to the best of our knowledge , there is no study investigating rt measurements in keratoconic corneas . compared rt , gat and dct for measuring iop in 171 eyes with corneal abnormalities . they reported that rt significantly underestimated iop according to gat ( 12.7 mm hg vs 15.5 mm hg ) measurements . however , only 21 eyes of 171 eyes had keratoconus in this study , being post - keratoplasty eyes ( 95 of 171 ) the most relevant of the corneal abnormalities included in this study . arribas - pardo et al . compared iop measurements in 60 ectatic corneas having intrastromal corneal ring segment implantation using the rt and gat . they found that icare significantly underestimated iop compared with gat , whereas icare pro showed no statistical differences compared with gat . we found that rt measurements were on average 2.38 mm hg lower than gat measurements . rt more affect than gat from cct in this study ( see table 3 ) , therefore individuals had thin corneas such as keratoconus iop tended to be underestimate with rt . dct is a method to measure iop by using a pressure - sensitive tip that is closely shaped following the corneal curvature to minimize the corneal deformation . the forces of both sides of the cornea are meant to be nearly equal during the measurement . according to studies using human cadaver eyes , iop values measured by dct were significantly closer to the manometric reference pressure than the gat measurements . previous studies have demonstrated an excellent agreement between gat and dct , although dct readings tended to be generally higher in healthy eyes and glaucomatous eyes.4 , 9 , 10 , 18 conversely to rt , there are many studies investigating dct measurements in keratoconic corneas . these studies are summarized in table 4.19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 dct measurements about 4 mm hg high than gat measurements in keratoconic corneas shown in table 4 . overestimation of dct measurements can be explained the absence of correlation of iop measurements with cct , especially in eyes with thinner corneal thickness just as keratoconic corneas . also gat measurements have been found to be higher than dct measurements in eyes with thicker corneas . in agreement with previous studies , we found that found that dct measurements were 3.70 mm hg higher than gat measurements in our study.today , many studies have shown that cct is variable and is a major source of error in gat , so the common practice of relying on unadjusted gat results in misdiagnosis and mismanagement.1 , 12 we considered two corneal parameters ( cct and cr ) to evalute their effect on iop measurements in this study . we found significantly positive correlation between cct and iop measurements in gat and rt but not in dct . rt is the most affected tonometer and dct is the least affected tonometer from cct . similarly , our previous study has reported that the iop measured by rt increased 8 mm hg for every 100-micron increase in cct . on the other hand , there is no consensus in the literature about the relation between the cct and iop measurements by rt ; some of the studies state that cct affect the iop measurements by rt5 , 6 , 28 but the others did not report this relationship.7 , 29 dct did not significantly correlate with cct in our study . dct has been proposed to measure iop irrespective of the corneal thickness because dct does not flatten the cornea , which allows the cornea to maintain its shape provoking minimal distortion during the measurement.4 , 8 , 9 , 10 , 16 , 17 , 18 many published studies have suggested correction factors based on cct and gat , but the effects of cr on iop measurements by gat remain uncertain . orssengo and pye discussed the deformation of a central cornea flattened by the pressure of the prism and bulging outward from the middle to the peripheries due to the inner pressure of the eye . however , other studies could not find any significant correlation between cr and iop.32 , 33 gunvant et al . reported that an increase of 1 mm of mean cr was accompanied by a rise in iop of 1.14 mm hg measured by gat , but this effect was weak and not statistically significant . our previous study show that cr had no significant effect on measured iop with rt in 165 healthy schoolchildren . francis et al . reported that cr affected iop measurements with dct but not with gat , with mean dct iop increased with increasing cr . we found a positive significant correlation between cr and iop measurements in rt but not in gat and dct in this study . the ocular response analyzer was the first device introduced to measure biomechanical features of the cornea . corneal resistance factor has been formulated to delineate the effect of cct on corneal hysteresis . both corneal hysteresis and corneal resistance factor decrease in keratoconic corneas compared with normal patients , which indicates mechanical weakening of the stroma.23 , 37 , 38 we could not evaluate corneal biomechanics in this study , and it may be pointed as the limitation of our study . sahebjada et al . reported that corneal thickness at the center and at the apex is different in different levels of keratoconus severity . these changes result in significant alterations in these parameters as severity of the disease increases . in addition to this , corneal pachymetric readings correlate well with corneal curvature but cct and the cr may not match with the thinnest point of the cornea or the radius of curvature in the area of the conus . in conclusion , dct has overestimated but rt has underestimated iop readings according to gat measurements in keratoconic corneas . we also found significantly positive correlation between cct and iop measurements in gat and rt but not in dct . f performed study concept and design , literature search , patient 's randomization and data collection , writing and reviewing of the study . ny performed study concept and design , literature search , writing and reviewing of the study .

backgroundmanagement options for pancreatic neuroendocrine tumors ( pnets ) metastatic to the liver include surgical , ablative , cytotoxic , and radioisotope approaches . one potential local treatment option includes selective internal radiotherapy utilizing yttrium-90 ( 90y ) microspheres . 90y has also been used in the treatment of hepatocellular carcinoma and tumors metastatic to the liver . it appears to be well tolerated ; however , there is no randomized controlled trial reporting long - term toxicities . previous retrospective reports have described biliary damage as a potential complication of therapy with 90y and chemoembolization ; however , the long - term sequelae of 90y treatment are poorly understood.case presentationwe present the case of a 65-year - old caucasian woman who suffered biliary damage following 90y administration for metastatic pnets and subsequently developed cirrhosis . given the timeline of her various treatments and the lack of any other identifiable etiology for her cirrhosis , we believe this to be a potential long - term complication of 90y therapy.conclusionthis case provides pathologic confirmation of cirrhosis as a potential long - term sequela of 90y treatment . this long - term risk needs to be considered when sequencing therapy for patients with neuroendocrine tumors who have a good prognosis . there are now several other systemic and ablative treatment options available to these patients , and long - term complications must be considered during treatment . purposeto compare the intraocular pressure ( iop ) measurements obtained with the rebound tonometry ( rt ) , dynamic contour tonometry ( dct ) and goldmann applanation tonometry ( gat ) in keratoconic corneas and to investigate the effects of central corneal thickness ( cct ) and corneal radius of curvature ( cr ) on iop measurements.methodssixty-three eyes of 63 keratoconus patients were enrolled in this cross - sectional study . iop was measured on each subject always in the same order , icare rt - pascal dct - gat , after a minimum interval of 10 min between measurements . cct and cr were measured using a rotating scheimpflug camera before the iop measurements in all subjects . one way repeated measures anova and pearson correlation coefficient analysis was used for the statistical assessment.resultsmean iop for all enrolled eyes was 11.72 2.59 mm hg for gat , 9.34 3.29 mm hg for rt , and 15.42 3.31 mm hg for dct . there were statistically significant differences among the three tonometers ; gat and rt ( p < 0.001 ) , gat and dct ( p < 0.001 ) , and rt and dct ( p < 0.001 ) . gat and rt were significantly positively correlated with cct ( r = 0.288 , p = 0.025 and r = 0.483 , p < 0.001 , respectively ) . rt was also significantly positively correlated with cr ( r = 0.550 , p < 0.001 ) . dct was not significantly correlated with cct ( r = 0.115 , p = 0.377 ) nor cr ( r = 0.179 , p = 0.168).conclusionsdct has overestimated but rt has underestimated iop readings according to gat measurements in keratoconic corneas . dct may be the most appropriate tonometer to use in keratoconus for the measurements of iop , because dct do not appear to be dependent upon cct and cr .

the other types of internal hernia that have been described include transmesenteric , supra- and/or perivesical , intersigmoid , foramen of winslow , and rarely , omental hernias . paraduodenal hernia is usually a congenital anomaly due to non - fusion of the mesocolon with the parietal wall that leaves a potential space . it manifests commonly in adults in the age group of 5060 years and usually presents as intestinal obstruction . internal hernias in todays era of advanced laparoscopic surgery are usually due to rents or defects in the mesentery . we present a case of intestinal obstruction due to internal herniation following laparoscopic donor nephrectomy , followed by a brief discussion . a 47-year - old lady presented with a history of vomiting and constipation for the past 5 days . upper gastrointestinal ( gi ) endoscopy revealed large amount of bile stained fluid in the stomach . a cect suggested dilatation of the stomach , duodenum and proximal jejunum with features of bowel obstruction in the mid jejunum , with the loops lying in left renal fossa [ figure 1 ] . patient was explored laparoscopically , through the same port sites that were made earlier for the nephrectomy ( two 10 mm ports , one at the level of umbilicus on the left side lateral to rectus muscle and another in the midclavicular line just above the left anterior superior iliac spine ; two 5-mm ports , one subcostal in the midclavicular line and another in the anterior axillary line in the lumbar region ) . herniation of small bowel was noted in the left paraduodenal space [ figure 2 ] . the herniation had occurred through a defect in the transverse mesocolon which probably would have occurred during the donor nephrectomy procedure . the loop of bowel was densely adhered to the tail of the pancreas and had to be dissected of it and reduced [ figure 3 ] . patient had an uneventful recovery and was discharged from hospital on the 5 postoperative day . cect scan showing obstructed bowel loops in the left renal fossa image showing herniated bowel loops image showing obstructed bowel loops being separated from the tail of pancreas image showing the end result following the closure of the mesocolic defect the increased use of laparoscopy for colonic resections , bariatric surgery , specifically the procedure of gastric bypass and laparoscopic nephrectomies have resulted in an increase in the postoperative complication of intestinal obstruction due to internal hernias . internal hernias have also been reported after liver transplantation specifically where the biliary continuity is restored through a roux - en - y choledochoenteric anastomosis . these hernias usually occur through the mesenteric or mesocolic defects that are either deliberately or inadvertently made during the above mentioned procedures and are not subsequently closed . in view of the fact that internal herniation of small bowel occurs through the transmesenteric or transmesocolic defects , it is generally recommended that these rents or openings should be specifically sutured and closed to prevent postoperative intestinal obstruction . in a retrospective review of 1,064 cases of laparoscopic gastric bypass , comeau and colleagues strongly recommended complete closure of all mesenteric defects to avoid internal hernias and their associated complications . however , following laparoscopic right colectomy for neoplasia in 530 consecutive patients , over a 7-year period , cabot et al . the incidence of intestinal obstruction was not statistically significant in their series when these transmesenteric defects were not sutured . they , however , stressed the need for more extended long - term follow - up in these series . most publications dealing with internal hernias following laparoscopic procedures in todays age do insist on closing mesenteric defects.[58 ] the diagnosis of intestinal obstruction due to internal herniation is usually difficult to be made by clinical signs and symptoms . plain radiographs as well as contrast - enhanced ct may sometimes suggest upper small gut obstruction . cect usually shows the jejunum massed in the left renal fossa as was reported in our case . though our case was not a true congenital paraduodenal hernia , the internal herniation had occurred into the left paraduodenal space through the mesocolic defect . surgical management of internal hernias has been traditionally performed by laparotomy , reduction of the hernia contents and closure of the defect . subsequently , laparoscopic repairs , using mesh as well as meshless have been performed by many groups . intestinal obstruction due to internal hernias following laparoscopic radical nephrectomy and laparoscopic donor nephrectomy , though few , have been reported in the literature . management in most of these cases has been exploration through either a laparotomy or laparoscopy . if the entrapped bowel is viable and not gangrenous , it can be usually reduced laparoscopically and the mesenteric or mesocolic rent sutured subsequently , as was done in our case . laparoscopic donor nephrectomy is the procedure of choice today , for procurements of kidneys across many centres , worldwide . small bowel obstruction due to internal hernia following this procedure though rare can lead to significant morbidity in an otherwise normal and healthy person . we do stress the need to be vigilant while mobilizing the colon and to suture any mesenteric or mesocolic defect deliberately or inadvertently made during the procedure . the other aspect to be highlighted is that in the current era , laparoscopy can be the modality of management especially in those patients who are haemodynamically stable and there is no suggestion of bowel necrosis or gangrene . omega-3 ( n-3 ) fatty acids can be divided into alpha - linolenic acid ( ala ; c18:3n-3 ) from plant origin , and eicosapentaenoic acid ( epa ; c20:5n-3 ) and docosahexaenoic acid ( dha ; c22:6n-3 ) from seafood . because the human body lacks the enzymatic capacity to synthesize ala the estimated average ala intake in the united states and most european countries is 1.3 to 1.7 g / d [ 13 ] . the institute of medicine ( iom ) of the national academies established dietary reference intakes for macronutrients in 2002 . for ala , the adequate intake ( ai ) was set at 1.6 g / d for men and 1.1 the iom noted that intakes of n-3 fatty acids above the ai may confer additional health benefits , especially with respect to cardiovascular health . many advisory boards consider ala intakes greater than 1.5 g / d important for human health . to achieve an adequate ala intake , food sources such as flaxseed and flaxseed oil , walnuts and walnut oil , and canola and soybean oil are recommended . for epa and dha from fish this evidence has been confirmed in randomized controlled trials [ 58 ] . for ala , several large , prospective cohort studies in the united states have shown inverse associations of ala intake with risk of cardiovascular diseases , but other epidemiologic studies have been inconclusive [ 911 ] . a meta - analysis of observational studies showed that increased intake of ala might reduce coronary heart disease ( chd ) mortality by 21% , although this was not statistically significant . in the lyon diet heart study , a randomized controlled trial in coronary patients , consumption of a mediterranean - type diet that included an additional daily intake of roughly 1 g of ala significantly decreased the risk of cardiac death and nonfatal myocardial infarction ( mi ) by more than 60% . this study , however , was not specifically designed to assess the effect of ala supplementation , and many dietary factors differed between the experimental and control group . since then , no randomized controlled trials of ala and cardiovascular events have been published . this article summarizes the current literature ( published after 2008 ) on dietary ala intake , ala tissue levels , and cardiovascular health in humans . several trials have recently been published on the effect of ala supplementation on blood lipids , inflammatory markers , and other indicators of cardiovascular health ( table 1 ) [ 13 , 14 , 1517 , 18 ] . trials were mostly of relatively short duration ( 612 weeks ) and ala doses ranged from 1.2 to 3.6 g / d . there was one long - term trial ( 52 weeks ) in which a high ala dose of 8.8 g / d was given [ 18 ] . increased ala intake was achieved by means of flaxseed oil [ 13 , 15 ] , ala - enriched margarine [ 14 ] , or other ala - enriched foods [ 16 , 17 , 18 ] . table 1overview of randomized controlled trials of increased alpha - linolenic acid intake and cardiovascular risk factors published between january 2008 and june 2010study / yearpopulationdesignoutcome for alakaul et al . /200888 healthy non - smoking canadian men and premenopausal women , aged 33 y12-week , double - blind , parallel randomized controlled trial ; sunflower oil ( placebo ) , flaxseed oil ( 1 g / d ala ) , hempseed oil ( 0.3 g / d ala ) , or fish oil ( 0.6 g / d epa+dha)plasma ala levels increased ( p < 0.05 ) after 6 wk ; no differences in total cholesterol , ldl - c , hdl - c , tg , ldl oxidation , platelet aggregation , or inflammation markers ( crp , tnf-)egert et al . [ 14]/200979 healthy non - smoking german men and premenopausal women , aged 1945 y6-week , double - blind , parallel randomized controlled trial ; ala ( 3.4 g / d ) , epa ( 2.2 g / d ) , or dha ( 2.3 g / d ) via enriched margarinesldl - ala levels increased ( p < 0.05 ) ; fasting serum tg decreased ( p < 0.05 ) ; no differences in total cholesterol , ldl - c , or hdl - cbarcel - coblijn et al . /200862 american men > 40 y of age12-week , parallel randomized controlled trial ; different doses of flax oil , fish oil , and sunflower oil in capsules ; ala doses were 1.2 g / d , 2.4 g / d , and 3.6 g / d2.4 and 3.6 g / d of ala significantly increased erythrocyte ala and epa levels ; no differences in inflammation markers ( crp , tnf- , svcam-1 ) , total cholesterol , tg , or hdl - csioen et al . /200959 healthy belgian male prisoners12-week single - blind study ; diet with 3.2 g / d extra alano effect on waist circumference , weight , bmi , systolic blood pressure ; diastolic blood pressure decreased and hdl - c increased in non - smokersbloedon et al . /200862 men and post - menopausal women from philadelphia , aged 4475 y , with hypercholesterolemia10-week , blind , parallel randomized controlled trial with low - fat diet with extra flaxseed or with wheat bran ( control ) ; ala dose of 3.4 g / dserum ala levels increased ; ldl - c was decreased after 5 wk but not after 10 wk ; lipoprotein(a ) was decreased and insulin sensitivity ( homa index ) improved ; no effect on inflammation ( il-6 , hs - crp ) or oxidative stress ( ox - ldl , urinary isoprostane ) ; hdl - c was reduceddodin et al . [ 18]/2008199 canadian menopausal women , aged 4965 y52-week , blind parallel trial ; 40 g / d flaxseed or wheat germ ; ala dose of 8.8 g / dserum ala levels increased ; modest effects on apolipoproteins a - i and b ; no effects on ldl electrophoretic characteristics or markers of hemostasis and inflammationala alpha - linolenic acid ; bmi body mass index ; dha docosahexaenoic acid ; epa eicosapentaenoic acid ; hdl - c high - density lipoprotein cholesterol ; homa homeostasis model assessment ; hs - crp high - sensitivity c - reactive protein ; il-6 interleukin-6 ; ldl - c low - density lipoprotein cholesterol ; svcam-1 soluble vascular cell adhesion molecule-1 ; tg triglycerides ; tnf tumor necrosis factor overview of randomized controlled trials of increased alpha - linolenic acid intake and cardiovascular risk factors published between january 2008 and june 2010 ala alpha - linolenic acid ; bmi body mass index ; dha docosahexaenoic acid ; epa eicosapentaenoic acid ; hdl - c high - density lipoprotein cholesterol ; homa homeostasis model assessment ; hs - crp high - sensitivity c - reactive protein ; il-6 interleukin-6 ; ldl - c low - density lipoprotein cholesterol ; svcam-1 soluble vascular cell adhesion molecule-1 ; tg triglycerides ; tnf tumor necrosis factor kaul et al . studied the effects of low - dose supplementation ( 2 g / d , by means of capsules ) of flaxseed oil , fish oil , and hempseed oil in 86 healthy canadian men and women about 34 years of age in a 12-week , randomized , double - blind , placebo - controlled trial . flaxseed oil ( ala dose 1 g / d ) increased plasma ala by 30% to 40% , had a small nonsignificant effect on plasma epa ( 7%8% ) , and no effect on plasma dha . none of the treatments affected serum total , low - density lipoprotein ( ldl ) , or high - density lipoprotein ( hdl ) cholesterol , triglycerides , ldl oxidation , platelet aggregation , or inflammatory markers . in another randomized trial in 62 healthy men approximately 40 years of age in the united states , participants were randomized to flax oil ( ala doses of 1.2 , 2.4 , or 3.6 g / d ) , fish oil ( 0.6 or 1.2 g / d ) , or sunflower oil for 12 weeks . increasing doses of flax oil caused elevation of the ala content in erythrocyte membranes by 40% to 80% , and also of epa ( 20%35% ) , but not of dha . none of the treatments altered plasma inflammatory markers ( c - reactive protein [ crp ] , tumor necrosis factor [ tnf]- , soluble vascular cell adhesion molecule-1 [ svcam-1 ] ) , plasma total or hdl cholesterol , or triglycerides . enrolled 62 american men and post - menopausal women aged 44 to 75 years with hypercholesterolemia . all participants were on a low - fat , low - cholesterol diet and subsequently randomized to flax - based or wheat - based products for 10 weeks . ala intake in the flaxseed group was 3.4 g / d higher than in the control group . flax - based products improved insulin sensitivity and reduced plasma ldl cholesterol by 7% to 13% and lipoprotein(a ) by 14% compared with wheat - based products . in men , there was also a decrease in hdl cholesterol . treatment had no effect on markers of inflammation ( interleukin-6 , high - sensitivity crp ) or oxidative stress ( oxidized ldl , urinary isoprostanes ) . the extent to which the observed effects of the flax - based diet were due to its ala content or other components of flax seeds is not clear . in a single - blind , 12-week field study among 59 healthy male prisoners ( mean age of 42 years ) in belgium , daily ala intake was increased from 2.8 to 4.9 g / d by means of ala - enriched foods without changing the linoleic acid content of the diet . all individuals were first on a regular diet , which was followed by an ala - rich diet , and no randomization was applied . epa and dha , but not ala , were assessed in platelet phospholipids , which showed no significant changes during ala intervention . body weight , waist circumference , and systolic blood pressure did not change , and there were no effects of ala on plasma total and ldl cholesterol , triglycerides , apolipoproteins a - i and b , glucose , and crp . [ 14 ] studied the effects of increased intake of ala , epa , and dha in a 6-week parallel trial in 74 german normolipidemic men and women aged 19 to 43 years . participants were randomly assigned to trial margarines that provided additional ala ( 3.4 g / d ) , epa ( 2.2 g / d ) , or dha ( 2.3 g / d ) . no placebo group was included . in the ala group , the ala content of ldl particles increased by 178% and the epa content increased by 36% , whereas dha remained unchanged . fasting serum triglycerides significantly decreased with epa ( 0.14 mmol / l ) , dha ( 0.30 dha intake significantly increased serum hdl cholesterol , whereas no changes were found with ala or epa intake [ 14 ] . finally , dodin et al . [ 18 ] evaluated the effect of flaxseed on markers of cardiovascular disease risk in 169 healthy menopausal canadian women who were randomly assigned to 40 g / d of flaxseed - based products or wheat - based products for 12 months . in the active - treatment group , flaxseed products increased plasma ala by 85% and plasma epa by 51% after 12 months of intake , which differed significantly from wheat - based intervention ( 12% and 29% , respectively ) . the 12-month intervention with flaxseed had significant effects on body weight ( 0.8 kg ) and serum total cholesterol ( 0.20 ldl cholesterol was reduced by 0.13 mmol / l ( p = 0.09 ) . apolipoproteins a - i and b increased in both groups , but less during flaxseed - based than wheat - based intervention . treatment had no effect on plasma lipoprotein(a ) , fibrinogen , crp , insulin , glucose , or ldl peak particle size . zatnski et al . examined trends in chd mortality in 11 eastern european countries and linked these figures to national data on vegetable oil consumption after 1990 . they showed the strongest decline in chd mortality in countries where rapeseed oil ( 9% ala ) rather than sunflower oil ( 0% ala ) was used . although these data are suggestive for a beneficial effect of ala , no definite conclusions can be drawn because the countries also differed in many other aspects that could impact cardiovascular health . cross - country comparisons based on aggregate data can not be adjusted for potential confounders , ( eg , socioeconomic status , lifestyle and other dietary components ) and should be considered for hypothesis generation only . in costa rica , soybean oil has been substituted for palm oil since the 1980s , which has led to an increase in ala intake . [ 19 ] performed a case control study in 3638 costa rican individuals to examine the association between ala and nonfatal mi ( fig . 1 ) . ala intake was assessed by a 135-item food frequency questionnaire and ranged from 1.1 to 2.4 g / d ( mean , 1.6 g / d ) in this population . dietary ala was inversely associated with nonfatal mi , with odds ratios indicating a 39% reduced risk for approximately a 0.6-g / d difference in intake . the relationship between ala and mi was nonlinear and mainly confined to the lowest levels of intake . dietary ala intake in this study correlated well with ala in adipose tissue , plasma , and erythrocytes , but poorly with biomarkers of epa and dha , suggesting a direct cardioprotective effect of ala rather than via conversion to long - chain n-3 fatty acids [ 19 ] . 1odds ratios and 95% confidence intervals for nonfatal myocardial infarction ( mi ) by deciles of alpha - linolenic acid in adipose tissue ( a ) or intake ( b ) in a case control study of 3638 men and women in costa rica . data were adjusted for smoking status , physical activity , household income , history of diabetes mellitus , history of hypertension , waist - to - hip ratio , saturated fat intake , and linoleic and trans fatty acids in adipose tissue . [ 19 ] ; with permission ) odds ratios and 95% confidence intervals for nonfatal myocardial infarction ( mi ) by deciles of alpha - linolenic acid in adipose tissue ( a ) or intake ( b ) in a case control study of 3638 men and women in costa rica . data were adjusted for smoking status , physical activity , household income , history of diabetes mellitus , history of hypertension , waist - to - hip ratio , saturated fat intake , and linoleic and trans fatty acids in adipose tissue . [ 19 ] ; with permission ) an anti - inflammatory action of ala has been proposed as an explanation for the inverse association with cardiovascular diseases found in the case control study in costa rica [ 19 ] , although negative findings on ala and inflammatory markers have been reported by others . [ 20 ] examined habitual ala intake and plasma concentrations of inflammatory markers , including il-6 and sil-6r , in 353 middle - aged men in the united states who were recruited from a twin registry . ala intake , assessed by the willett questionnaire , ranged from 0.2 to 2 g / d , and was significantly inversely related to plasma sil-6r independent of shared genetic factors and a wide range of potential confounders . a twin with a 1-g higher ala intake ( equivalent to one tablespoon of canola oil ) had 11% lower sil-6r concentrations than his twin with a low intake . despite the robust association with sil-6r , no significant associations were found with plasma il-6 , tnf- , or high - sensitivity crp [ 20 ] . in a study of 511 japanese employees of municipal offices , ala intake was assessed by a diet history questionnaire that had been validated against 16-day weighed food records ( pearson correlation for ala , r = 0.3 ) . ala comprised 0.8% to 1.0% energy ( > 2 g / d ) and was inversely related to serum crp . data were adjusted for age , body mass index ( bmi ) , lifestyle factors , and physical activity , but not for dietary factors associated with ala , and should therefore be interpreted with caution . [ 22 ] assessed dietary ala intake by the willett questionnaire in 260 post - mi patients and linked these data to 24-hour electrocardiogram recordings that were analyzed for ventricular premature beats . ala intake ( expressed per 1,000 kcal / d ) was associated with a significant reduction in ventricular premature beats after adjustment for age , sex , cardiovascular medication , and co - morbidities . however , lifestyle ( eg , smoking ) and dietary factors were not controlled for and the study had a cross - sectional design , which has inherent methodologic weaknesses . previous analyses in the national heart , lung , and blood institute nhlbi family heart study suggested that ala may protect against atherosclerosis [ 23 , 24 ] . recently , sala - vila et al . investigated whether serum phosphatidylcholine content of ala and other fatty acids was related to carotid atherosclerosis in a cross - sectional study of 451 asymptomatic spanish individuals ( mean age of 45 years ) with primary dyslipidemia . over half of the participants the habitual mediterranean diet in this population provided a mean ala intake of 0.8 g / d , mainly from olive oil and walnuts , which correlated well with serum ala ( r = 0.44 ) . the association of serum fatty acids with intima - media thickness ( imt ) of the carotid arteries was assessed , adjusting for age , sex , bmi , smoking , antihypertensive drugs , statins , and other serum fatty acids . significant inverse associations were found between serum ala and internal carotid artery imt , and between serum dha and common carotid artery imt . serum epa was not associated with imt . in a small cross - sectional study of 50 asian men and women ( mean age of 58 years ) who suffered a first nonfatal mi , the mean common carotid imt was inversely associated with ala content of erythrocytes ( p = 0.09 ) and ala intake ( p = 0.02 ) . data , however , were only adjusted for age , sex , and total energy ( for dietary ala ) . the average intake of ala was 0.6 g / d , which was similar to that of epa and dha intake . ebbesson et al . examined whether ala content of erythrocytes was related to heart rate as a risk indicator for ventricular arrhythmias . they performed a cross - sectional study in 707 alaskan eskimos ( mean age of 50 years ) who had a habitual intake of marine n-3 fatty acids of 2.9 g / d . after adjustment for gender , height , bmi , blood pressure , smoking , and heart rate lowering medications , no association was observed with ala ( p = 0.98 ) . epa and dha , on the other hand , were significantly inversely associated with heart rate . the delta(6)-desaturase enzyme is the rate - limiting step in the conversion of ala into epa and dha , and genetic variation in the delta(6)-desaturase gene ( fads2 ) may therefore affect the associations of ala with cardiovascular health . truong et al . , in a cross - sectional study , examined the association of ala in adipose tissue with metabolic syndrome , and possible effect modification by fads2 . in a cohort of 1,815 men and women from costa rica , the prevalence of metabolic syndrome ( 656 cases ) was 19% lower in the upper compared with the lower quintile of adipose tissue ala . there was no association between ala and metabolic syndrome among homozygous carriers of the fads2 deletion allele , suggesting that conversion of ala into epa may play a role . it should be noted , however , that the fads2 polymorphism did not influence the inverse association of ala with nonfatal mi in a previous case ala status has been inversely associated with cardiovascular disease events , although data are less consistent than for epa and dha [ 710 ] . an overview of ala biomarker studies is provided in table 2 . in the aforementioned case [ 19 ] , ala was measured in adipose tissue as a biomarker of intake , which correlated well with dietary ala . ala was strongly inversely associated with nonfatal mi , with a 57% reduced risk when comparing the 7th decile with the lowest decile . the relationship between ala and mi was nonlinear and mainly confined to levels below the median ( fig . 1 ) . mozaffarian et al . suggested that ala may particularly reduce chd risk when intake of marine n-3 fatty acids is low . in the study by campos et al . [ 19 ] , however , concurrent fish intake ( range , 332 g / d ) or epa and dha intake ( range , 130520 mg / d ) did not modify the associations of ala with mi . table 2overview of biomarker studies of alpha - linolenic acid and cardiovascular events published between january 2008 and june 2010study / yearpopulationdesignoutcomecampos et al . [ 19]/2008costa rica : 1,891 cases with first nonfatal mi and 1,891 population - based controls ; matching for age , sex , and area of residencecase control study : association of ala intake from ffq and ala in adipose tissue with risk of first nonfatal mior ( 95% ci ) for first nonfatal mi was 0.41 ( 0.250.67 ) for top vs lowest decile of ala in adipose tissue , and 0.61 ( 0.420.88 ) for high vs low ala intake ; associations only present at lower ala levelsyamagishi et al . /2008usa , minneapolis : 3,575 white men and women from aric study , ages 4564 yprospective cohort study : association of plasma ala with incident heart failure ; 14.3 y of follow - up195 participants ( 5.5% ) developed heart failure ; ala status ( top vs bottom quintile ) was not associated with incident heart failure ; age- and sex - adjusted hr was 0.99 ( 0.631.53 ) for cholesteryl ester fraction and 0.97 ( 0.611.54 ) for phospholipid fractionwarensj et al . /2008sweden : 2,009 men from ulsam study , aged 50 yprospective cohort study : association of ala in serum cholesteryl esters with cvd mortality ; 30.7 y of follow - upmultivariable - adjusted hr was 1.10 ( 1.001.21 ) per 1-sd increase in serum alapark et al . /2009south korea : 40 cases of ischemic stroke , 40 cases of hemorrhagic stroke and 40 healthy controls ; matching for age and sexcase control study : association of ala in erythrocytes with risk of ischemic and hemorrhagic strokeerythrocyte ala concentrations ( area% ) in hemorrhagic stroke patients ( 0.71 0.21 ) and ischemic stroke patients ( 0.24 0.03 ) were not significantly different from controls ( 0.44 0.05 ) after adjustment for family history of stroke ; inverse association of ala with ischemic stroke after adjustment for age and systolic blood pressure ( p = 0.045).virtanen et al . [ 34]/2009finland : 2,174 men from kuopio ischemic heart disease risk factor study , ages 4260 yprospective cohort study : association of serum ala with incident atrial fibrillation ; 17.7 y of follow - up240 men ( 11.0% ) developed atrial fibrillation ; multivariable - adjusted hr for serum ala ( compared to q1 ) was q2 : 1.26 ( 95% ci , 0.841.89 ) , q3 : 0.74 ( 0.461.20 ) , and q4 : 1.14 ( 0.721.79 ; p for trend = 0.98).lemaitre et al . [ 35]/2009usa , seattle : 265 out - of - hospital sudden cardiac arrest patients and 415 community members ; matching for age , sex , and calendar yearcase control study : association of ala in erythrocytes with risk of sudden cardiac death ; blood collection immediately after the event ( patients ) or during interview ( control)multivariable - adjusted or over quartiles of ala in erythrocytes ( compared to q1 ) : q2 was 1.7 ( 1.03.0 ) , q3 was 1.9 ( 1.13.3 ) , q4 was 2.5 ( 95% ci , 1.34.8 ) ; association independent of erythrocyte levels of epa and dha , linoleic acid , and trans fatty acidsala alpha - linolenic acid ; aric atherosclerosis risk in communities ; cvd cardiovascular diseases ; dha docosahexaenoic acid ; epa eicosapentaenoic acid ; ffq food frequency questionnaire ; hr hazard ratio ; mi myocardial infarction ; or odds ratio ; q quartile ; sd standard deviation ; ulsam uppsala longitudinal study of adult men overview of biomarker studies of alpha - linolenic acid and cardiovascular events published between january 2008 and june 2010 ala alpha - linolenic acid ; aric atherosclerosis risk in communities ; cvd cardiovascular diseases ; dha docosahexaenoic acid ; epa eicosapentaenoic acid ; ffq food frequency questionnaire ; hr hazard ratio ; mi myocardial infarction ; or odds ratio ; q quartile ; sd standard deviation ; ulsam uppsala longitudinal study of adult men yamagishi et al . the data used were from 3,575 white men and women in the united states ( mean age of 54 years ) who participated in the atherosclerosis risk in communities ( aric ) study . during 14 years of follow - up plasma ala was not associated with risk of heart failure ( hazard ratios of 0.99 and 0.97 for upper versus lower quintiles of cholesteryl ester and phospholipid fractions , respectively ; p = 0.8 ) . data were extensively adjusted for major confounders , including age , sex , bmi , lifestyle factors , and cardiovascular risk factors , but not for potential dietary confounders . conducted a prospective cohort study of ala in serum cholesteryl esters and cardiovascular mortality in more than 2,000 swedish men ( mean age of 50 years ) . the study had 30 years of follow - up and comprised over 60,000 person - years . a 10% higher risk of fatal cardiovascular events was found per standard deviation increase in serum ala , which was borderline statistically significant . data were adjusted for serum cholesterol , bmi , smoking , physical activity , and presence of hypertension . erythrocyte concentrations of n-3 fatty acids were studied in relation to hemorrhagic and ischemic stroke in a case control study of 120 asian men and women ( mean age of 57 years ) . the average total n-3 fatty acid intake in this population was 1.1 g / d , about half of which was ala . erythrocyte ala concentrations ( area% ) were 0.71 0.21 in hemorrhagic stroke patients and 0.24 0.03 in ischemic stroke patients , which did not differ significantly from controls ( 0.44 0.05 ) after adjustment for family history of stroke . in a logistic regression model , both epa and dha were also inversely related to hemorrhagic stroke , but this was not the case for ala . data , however , were only adjusted for age and systolic blood pressure , so residual confounding may have been present . the risk of atrial fibrillation in relation to serum n-3 fatty acids was examined in a prospective cohort study of 2,174 finnish men ( mean age of 53 years ) [ 34 ] . during 18 years of follow - up hazard ratios in consecutive quartiles of ala were 1.26 ( 95% ci , 0.841.89 ) , 0.74 ( 95% ci , 0.461.20 ) , and 1.14 ( 95% ci , 0.721.79 ) compared with the lowest quartile ( p for trend = 0.98 ) . serum epa did not show an association but dha was inversely related to atrial fibrillation ( hazard ratio of 0.62 for upper vs lower quartile ; p for trend = 0.02 ) [ 34 ] . [ 35 ] investigated in a case control study whether ala levels in erythrocytes were associated with risk of sudden cardiac death . blood was collected from 265 out - of - hospital sudden cardiac arrest patients in seattle , wa immediately after the event and from 415 randomly selected community members ( mean age of 58 years ) . in contrast to what was expected , higher ala levels were associated with a higher risk of sudden cardiac arrest . after adjustment for age , sex , smoking , diabetes , hypertension , education , physical activity , weight , height , and total fat intake , the risk increased over the quartiles with an odds ratio of 2.5 ( 95% ci , 1.34.8 ) for the highest compared with the lowest quartile . the association was independent of other fatty acids in erythrocyte membranes , including epa and dha [ 35 ] . the trials reviewed here consistently showed an increase in blood ala levels after ala supplementation , starting at low doses ( < 2 g / d ) . ala supplementation also increased blood levels of epa , but not of dha , indicating conversion of ala to epa through elongation and desaturation . short - term trials ( 612 weeks ) in generally healthy individuals mostly showed no or inconsistent effects of ala intake ( 1.23.6 g / d ) on blood lipids , ldl oxidation , lipoprotein(a ) , and apolipoproteins a - i and b. previous studies suggested an anti - inflammatory action of ala [ 5 , 7 , 8 ] , but recent trials showed little effect of ala on crp or other inflammatory markers . there was , however , an interesting inverse association of ala with sil-6r in the twin study by dai et al . the few studies that addressed ala intake in relation to glucose metabolism or blood pressure yielded inconsistent results [ 16 , 17 , 18 ] . there is , however , some observational evidence that a high ala status may be related to a lower risk of metabolic syndrome . long - term treatment with high ala doses had a beneficial effect on body weight and blood ldl cholesterol [ 18 ] , which warrants confirmation in future trials . 23 g / d ) for the primary and secondary prevention of chd . the recently published case control study by campos et al . [ 19 ] showing strong inverse associations of ala status and intake with nonfatal mi is in line with this recommendation . recent data provide some support that ala could protect against atherosclerosis [ 25 , 26 ] , but it should be noted that this evidence comes from relatively small cross - sectional studies and that conclusions on causality can not be drawn . concerning the hypothesis of an anti - arrhythmic effect of n-3 fatty acids , there was some evidence for protection against ventricular premature beats [ 22 ] , but no evidence for a relation with heart rate or incidence of atrial fibrillation [ 34 ] . moreover , data from two recent well - conducted epidemiologic studies suggested that high tissue ala is related to an increased rather than decreased risk of fatal cardiovascular events and sudden death [ 35 ] . performed a meta - analysis of previous studies on tissue fatty acid composition and risk of chd published until 2006 . they showed that ala in adipose tissue or phospholipids was inversely associated with chd , although the association was not statistically significant for fatal events . of 16 studies reviewed , about half were supportive for a beneficial role of ala , whereas other studies were negative or supportive for an adverse effect . in conclusion , there is a need for long - term trials investigating the effect of ala supplementation on cardiovascular risk factors and clinical end points . data are awaited from the alpha omega trial , in which 4,837 post - mi patients ( mean age of 69 years ) were randomized to 2 g / d of ala and/or 400 mg / d of epa and dha and followed for fatal and nonfatal cardiovascular disease events for 40 months . the findings of this study may answer the question of whether a food - based dose of ala affects cardiovascular health in high - risk individuals and , if so , whether this is comparable to the effect of marine n-3 fatty acids .

internal hernias are a rare cause of small bowel obstruction . following laparoscopic bariatric surgery , specifically gastric bypass and laparoscopic colonic resections , there has been an increase in the incidence of internal hernias . this has been due to either a mesenteric or mesocolic defect being not closed or completely missed . small bowel loops usually herniate through these defects and present as intestinal obstruction . internal hernia following laparoscopic donor nephrectomy is a rare complication . the need for presenting this case is the rarity of its occurrence , to stress the fact that following major abdominal laparoscopic surgery the mesenteric or mesocolic defects should be closed , and that this complication was managed laparoscopically , through the same port sites as used earlier for the donor nephrectomy . there is a large body of scientific evidence that has been confirmed in randomized controlled trials indicating a cardioprotective effect for omega-3 fatty acids from fish . for alpha - linolenic acid ( ala ) , which is the omega-3 fatty acid from plants , the relation to cardiovascular health is less clear . we reviewed the recent literature on dietary ala intake , ala tissue concentrations , and cardiovascular health in humans . short - term trials ( 612 weeks ) in generally healthy participants mostly showed no or inconsistent effects of ala intake ( 1.23.6 g / d ) on blood lipids , low - density lipoprotein oxidation , lipoprotein(a ) , and apolipoproteins a - i and b. studies of ala in relation to inflammatory markers and glucose metabolism yielded conflicting results . with regard to clinical cardiovascular outcomes , there is observational evidence for a protective effect against nonfatal myocardial infarction . however , no protective associations were observed between ala status and risk of heart failure , atrial fibrillation , and sudden death . findings from long - term trials of ala supplementation are awaited to answer the question whether food - based or higher doses of ala could be important for cardiovascular health in cardiac patients and the general population .

pancreatic cancer is the fourth leading cause of cancer death in the western world and shows the worst mortality among common malignancies with a 5-year survival rate of lower than 5% . the current standard of care includes surgical resection when appropriate and gemcitabine - based chemotherapy . recent data suggests the use of folfirinox as initial treatment of patients with metastatic disease who have a good performance status improves survival . single - agent gemcitabine is often administered to patients with advanced , metastatic pancreatic cancer . studies with gemcitabine have demonstrated a significant clinical response ( decreased pain , increased functional status ) even in the absence of a measurable tumor response . unfortunately , there is currently no standard second - line chemotherapy for metastatic pancreatic cancer . one of the current major challenges is the lack of validated markers for prediction of response to chemotherapy in the metastatic pancreatic cancer setting . a 49-year - old hispanic male , with a 25-pack year smoking history , moderate alcohol consumption and a family history reportedly noncontributory for gastrointestinal malignancies or liver disease , was found to have a biopsy - proven poorly differentiated ductal adenocarcinoma of the pancreas . the patient had a past medical history of gastritis ; pancreatitis ; new onset insulin - dependent diabetes mellitus ; depression , and anxiety . a diagnostic ct of the abdomen performed at an outside institution immediately prior to presentation to our center , revealed an infiltrative mass of the head of the pancreas measuring 4.6 3.5 cm in transverse and anteroposterior diameters and approximately 3.4 cm in longitudinal diameter , which was obstructing the common bile duct and pancreatic duct , infiltrating the peripancreatic and perivascular spaces and occluding the superior and mesenteric vein . the pathology report described a poorly differentiated pancreatic adenocarcinoma that had metastasized to the liver . the patient 's ca19 - 9 was 5,590 u / ml at presentation , and cea 2.8 u / ml ( both doubled in value over a period of approximately 2 weeks prior to presentation to our center ; table 1 ) . symptoms at presentation consisted of fatigue and intermittent abdominal pain , controlled by pain medications ( opioids ) . the patient also reported an approximately 79 kg weight loss over a period of 34 months ( weight at presentation 59.4 kg ) . a ct - guided biopsy of the anterolateral right lobe liver lesion confirmed metastatic pancreas cancer . the immunostains performed on paraffin sections were supportive of the above diagnosis : the tumor was positive for ck7 , ck20 and ca19 - 9 and negative for ttf1 . it was decided to pursue with chemotherapy consisting of a doublet gemcitabine and oxaliplatin ( gemox ) . the patient received gemcitabine at 850 mg / m / dose on day 1 and day 8 and oxaliplatin at 100 mg / m / dose on day 2 , every 21 days . the patient also received l - glutamine powder 15 g twice / day dissolved in protein shakes . other supplements were taken along with chemotherapy ( self - administered ) : herbal mixtures anamu ( cuba ) and brim ( ecuador ) . the first three cycles of chemotherapy were reasonably well tolerated without any significant side effects . the patient 's appetite and nutritional intake improved and weight gain was noted . mild hyperkalemia was observed , presumed to be due to increased potassium intake ( nutritional shakes ) . a pet / ct evaluation performed after 3 cycles of chemotherapy revealed decreased fdg uptake within the lesion in the head of the pancreas ( suv of 2.8 compared to 6.5 on previous examination ) and left adrenal gland ( suv 2.7 decreased from 3.8 ) , compatible with response to therapy . there was complete resolution of the focus of mildly intense fdg uptake in the anterior right lobe of the liver . new minimal fdg uptake was observed in the left lower lobe of the lung , presumed to be of inflammatory etiology . the ct scan of the abdomen showed overall decrease in size and volume of the pancreatic head lesion and of the tiny left adrenal nodule , as well as resolution of anterior right hepatic lesion consistent with response to therapy . the patient completed 8 cycles of chemotherapy with complete radiographic and excellent tumor marker response ( ca19 - 9 level decreased from 5,590 u / ml to 130 u / ml ) and minimal symptoms . two treatment delays / dose reductions due to neutropenia developed during the 6-month treatment period . at the 1-month evaluation , the ca19 - 9 level continued to decrease from 130 u / ml to 55 u / ml , markedly decreased from 5,590 u / ml prior to initiation of therapy . the ct scan of the chest , abdomen and pelvis , revealed no definite pancreatic masses or liver metastases . the evaluation performed two months later , after a treatment break , showed essentially stable findings , with the ca19 - 9 slowly trending upwards to a value of 125 u / ml . at another 2-month interval , the pet / ct scan showed mild interval disease progression in the pancreatic head surrounding the common bile duct stent and interval progression of multifocal hepatic metastatic disease in both liver lobes , which was also evidenced on ct scan . the radiographic changes were paralleled by the elevation of ca19 - 9 level from 125 to 7,730 u / ml . at this point in time the patient was in overall good health , with good appetite and an ecog ps of 0 . second - line therapy with gtx regimen was initiated consisting of the following dosages / schedule : gemcitabine 600 mg / m day 1 and day 8 , docetaxel 30 mg / m day 4 and day 11 , and capecitabine at 1,000 mg p.o . an evaluation performed after 3 cycles of the second - line chemotherapy program revealed the patient to be doing reasonably well with mild numbness and tingling noted in the hands and feet . he had lost approximately 1.8 kg in weight but otherwise remained in reasonably good health . the ca19 - 9 level had decreased to 493 u / ml ( from 7,730 u / ml prior to the initiation of gtx regimen ) . the restaging ct scan of the chest , abdomen , and pelvis performed at this visit showed interval response to therapy of multifocal hepatic metastatic disease . at a subsequent evaluation performed after 5 cycles of this therapy there was continued evidence of a response , with the ct scan revealing a decreased size of the pancreatic head mass . however , there was a mild increase in size of two previously noted liver lesions and one small lesion was unchanged in size from the previous examination . the ca19 - 9 continued to decline and reached the level of 155 u / ml after 5 cycles of therapy , consistent with an ongoing treatment response . treatment with gtx regimen continued and the patient completed 8 cycles with good overall tolerance . at this point imaging studies revealed stable disease with a slight increase in the ca19 - 9 level ( from 155 u / ml to 264 u / ml ) . three months later the patient presented for a follow - up evaluation with reportedly increased epigastric and right upper quadrant pain , increased flatulence and a 4-kg weight loss during the previous two months . . a restaging ct scan of the chest , abdomen and pelvis showed significant interval progression within the liver with a large heterogeneous 9-cm central mass centered at the hepatic hilum with near occlusion of the right portal vein and mild to moderate bilateral intrahepatic biliary ductal dilatation . the ca19 - 9 level had increased from 264 to 90,200 u / ml . the treatment management plan consisted of initiation of third - line chemotherapy with gemcitabine and nab - paclitaxel ( abraxane ) . the regimen dosages / schedule were as follows : gemcitabine 700 mg / m and nab - paclitaxel 70 mg / m , both administered on day 1 , 8 and 15 , every 28 days . cycle 1 of nab - paclitaxel and gemcitabine was reasonably well tolerated with moderately well controlled pain in the epigastric area ( reportedly , the patient did not follow his pain regimen as indicated ) . the patient was also seen by interventional radiology and was not considered to be a suitable candidate for chemoembolization at that point in time . in view of the excellent tolerance to the gemcitabine / nab - paclitaxel chemotherapy regimen , improvement in liver function tests and hematology parameters , a decision was made to increase the gemcitabine dose to 900 mg / m and the nab - paclitaxel dose to 100 mg / m . during this time the patient received hematological growth factor prophylaxis . after 3 cycles of gemcitabine / nab - paclitaxel repeat imaging and tumor markers again demonstrated evidence of a favorable response . the ca19 - 9 level was markedly decreased from 90,200 u / ml to 4,830 u / ml and the ct of chest / abdomen revealed less compression of the portal vein compared to the previous study . the patient subsequently continued treatment with this regimen at an outside facility , eventually experiencing progression of the disease process . in summary , and of considerable interest , our patient lived for a total period of 2 years after his initial diagnosis of metastatic pancreatic carcinoma . pancreatic cancer is one of the most lethal tumors of the gastrointestinal tract . the ability to predict which patients would benefit most from surgical intervention and/or chemotherapy would be a great clinical advance . considerable research has focused on identifying molecular events in pancreatic carcinogenesis , and the correlation with clinicopathological variables of pancreatic tumors and survival . for the most part , the evidence regarding the application of biomarkers as prognostic indicators in this malignancy is conflicting . the advent of gene microarray and mass spectrometric protein profiling offers the potential to examine many different biomarkers simultaneously . this work may allow researchers to develop accurate and reproducible predictors of survival based on genetic signatures . genetic and pharmacokinetic testing were not performed in our patient , but his exceptional response to chemotherapy treatments may lead to the conclusion that this patient 's pancreatic tumor may have had a unique genetic and molecular makeup . highlights of this case report are a dramatic clinical , radiographic and serologic ( tumor markers ) response observed with each chemotherapy regimen administered , as well as significant improvement of the performance status and quality of life for our patient , secondary to chemotherapy treatment . evidence supports the suggestion that treatment with gemcitabine / oxaliplatin is superior to gemcitabine alone in terms of response rate , clinical benefit rate and progression - free survival , but the differences did not reach the significant level for overall survival . data for second - line therapy with gemcitabine - based regimens after first - line gemcitabine treatment have been published . the combination of gemcitabine , docetaxel and capecitabine ( gtx ) is an active regimen in patients previously treated with gemcitabine for metastatic pancreatic cancer . better performance status and > 75% drop in pretreatment ca19 - 9 were associated with longer survival . gtx has a promising survival benefit of greater than 1 year in patients with advanced pancreatic adenocarcinoma when compared to studies using single agent gemcitabine or other combination chemotherapies . following second line with gtx our patient experienced a significant response to a third - line gemcitabine - based regimen ( gemcitabine and nab - paclitaxel ) . gemcitabine plus nab - paclitaxel has promising antitumor activity in unresectable and borderline resectable pancreatic cancer patients . pancreatic cancer cells and surrounding stroma are known to overexpress sparc ( secreted protein acid rich in cysteine ) , which is associated with poor clinical outcomes . nab - paclitaxel , an albumin - bound nanoparticle form of paclitaxel , increased tumor accumulation of paclitaxel through binding of albumin to sparc . a disease - specific phase 1 study was designed to evaluate the safety and efficacy of gemcitabine + nab - paclitaxel and the correlation of response with tumor sparc and serum ca19 - 9 levels . the combination of nab - paclitaxel and gemcitabine was generally well tolerated and had substantial enough antitumor activity in patients with pancreatic cancer to warrant a phase iii clinical trial . a number of possible hypotheses can be advanced for why our patient exhibited this unexpected degree of persistent sensitivity to gemcitabine - based chemotherapy , including an inability to repair chemotherapy - induced dna damage and a role for the glutamine supplementation he received during his treatment regimen . hopefully , future research in this area will elucidate the reasons for such relatively favorable outcomes . medication errors are known to be a significant source of risk and harm to pediatric patients [ 14 ] , particularly in neonatal and pediatric intensive care units ( picus ) [ 58 ] . a study looking at prescribing errors intercepted by pharmacists in a maternity and children 's hospital found the risk of prescribing errors to be fourfold higher in pediatric patients than in the maternity population ( 1,391 errors in 61,458 orders versus 644 errors per 119,333 orders ) . they found that intercepted errors in pediatric patients were most commonly related to dosing , with detection of doses 1.5- to 10-fold higher than recommended . finally , the use of intravenous ( iv ) medications has also been identified as a specific source of medical risk [ 10 , 11 ] . in response to the errors and harm associated with medication infusions , the adoption of a range of healthcare information technologies including computer order physician entry ( cpoe ) , bar coded medication administration , and smart infusion pumps has been recommended [ 1214 ] . while these technologies have demonstrated the capacity to decrease the frequency of errors [ 1517 ] , results are mixed in studies trying to document reduction in harm . while some have been able to show a decrease in mortality [ 18 , 19 ] , others have failed to demonstrate efficacy of the technology or reduction in harm [ 2026 ] . notably , others have uncovered associations between cpoe and new and unanticipated errors as well as harm [ 2730 ] . the same authors as the present paper conducted a study in 2007 measuring discrepancies between medication orders for infusions entered into a cpoe system and the medication ( or intravenous fluid ( ivf ) ) actually being infused at the bedside within a picu . rates of discrepancy were calculated and discrepancies were categorized by type . a high frequency of discrepancies between cpoe orders for medications and programmed settings on smart infusion pumps at the bedside ( 24% ) and between intravenous fluids and programmed pump settings ( 42% ) was observed . we concluded that the discrepancies were most likely related to interactions between providers performing the tasks of ordering intravenous medications and fluids and the programming of the infusion pumps and that more intensive understanding of these interactions was essential to the process of risk mitigation and elimination . since the time of the original study , a bidirectional interface between the cpoe and pharmacy systems was established at the study site . this allowed orders entered by providers to go directly to the pharmacy system for verification rather than being printed and reentered into the pharmacy system by a pharmacist prior to order verification . closed - loop medication management systems automate steps in the complex process of medication delivery in an effort to reduce the number of opportunities for mistakes . relatively little has been published about the performance of these systems . one study in 2012 demonstrated a decrease in time from order placement to administration of antibiotics following implementation of a closed - loop order - processing system . another study examined the effects of closed - loop electronic prescribing , automated dispensing , barcode patient identification , and electronic medication administration record and found a decrease in prescribing and administration errors . we were interested in examining the impact of a bidirectional interface between cpoe and pharmacy systems within a picu on the frequency and types of discrepancies between orders for medication and ivf infusions and settings on bedside infusion pumps . we hypothesize that the frequency of discrepancies will be less but that the observed changes in rates and types of discrepancies will not be attributable to the closed - loop system alone . the picu also underwent expansion and relocation in the interim , and we suspect that changes in workflow may be in part responsible for any observed decreases in discrepancies . the study was performed in a 72-bed picu with approximately 1800 admissions per year within a freestanding children 's hospital located in the midwest . patients admitted to this picu include children ranging from one day to over 18 years of age , with a wide range of disease processes including trauma , cardiothoracic surgery , respiratory failure , metabolic disease , and sepsis . orders for iv medication infusions and ivf may be entered into the cpoe system by attending physicians , trainees ( fellows and resident physicians ) , and nurse practitioners and physician assistants . data were gathered over a period of seventeen weekdays . between study periods , the picu also underwent relocation and expansion from 30 beds to 72 beds . the cpoe system , sunrise clinical manager version 4.5 by eclipsys technologies corporation , was implemented in september of 2000 a closed - loop system consisting of a bidirectional interface between cpoe and pharmacy systems was established in september of 2008 . intravenous medication infusions were defined as medications requiring the use of an infusion pump . only medications given as continuous iv infusion were included . medications given via infusion pump were excluded if dosing was intermittent , a change from methodology of the previous study . intravenous fluids were defined as total parenteral nutrition ( tpn ) , lipids , and crystalloid infusions given continuously via infusion pump . data were simultaneously collected from the medication orders in the cpoe system and the bedside infusion pumps by trained observers once a day over a period of seventeen days in august of 2010 . one observer recorded date , time , bed number , each infused medication or fluid , and corresponding dose or rate . a second observer simultaneously recorded existing orders by bed space , capturing the date and time of observation , and each ordered medication or fluid with its respective dose or rate . a line - by - line comparison of order observation data with the pump observation data was performed , matched by time and bed number . observations occurring more than fifteen minutes apart were excluded to minimize the effect of any interim changes to order or pump . for each observed medication infusion or ivf , analysis began with verifying the presence of a corresponding order . for those that had a corresponding order , the medication doses or ivf rates that were ordered were compared to those observed to be infusing at the bedside . discrepancies were defined as follows : a medication or fluid found to be infusing without a corresponding order was categorized as an unauthorized medication or unauthorized fluid . when no medication or fluid infusion was observed despite the presence of an active order , finally , when the medication or fluid infusing at the bedside was observed to differ in dose ( for medications ) or rate ( for fluids ) when compared to the active order in cpoe , this discrepancy was categorized as a wrong medication dose or wrong fluid rate . any difference in dose ( for medications ) or rate ( for ivf ) between the computerized order and the bedside pump setting proportions of discrepant medications and fluids for the two study periods ( original study in 2007 , current study in 2010 ) were compared . p values were calculated using two - proportion z test , and p values < 0.05 were considered significant . a total of 303 observations of medication infusions and 152 observations of ivf were completed during the study period . the number of medication observations was similar ( 296 observations in 2007 versus 303 observations in the current study ) . no observations required exclusion based on the predetermined 15-minute time window for orders and programming . during this study period 54 of 303 ( 18% ) observations of medication infusions revealed order - programming discrepancies , while 46 of the 152 ( 30% ) observations of ivf revealed order - infusion pump discrepancies ( tables 1 and 2 ) . the decrease seen in medication infusion discrepancies did not reach statistical significance ( p = 0.05 ) . however , this represented an overall decrease in the discrepancy rate for the ivf group , which included tpn , lipids , and crystalloid infusions , following implementation of the bidirectional interface ( p = 0.01 ) . the decrease in discrepancies between orders and ivf was due in large part to a significant decrease in discrepancies for tpn . ten of 55 ( 18% ) tpn observations had discrepancies , down from a rate of 50% in the prior study ( p < 0.01 ) ( table 2 ) . there were no significant changes in overall rates of discrepancy for crystalloid or intralipid infusions . however , the proportion of unauthorized fluid discrepancies ( ivf infusing without a corresponding order ) decreased significantly ( 32% to 15% , p < 0.05 ) ( table 3 ) . although the overall discrepancy rate for medications did not change following implementation of the pharmacy - order entry interface , the types of discrepancies that were observed did change . for example , unauthorized medications ( medications infusing without a corresponding order ) accounted for 60% of the medication discrepancies in the previous study and decreased to 4% ( p < 0.01 ) in the current study ( table 3 ) . on the other hand , we observed significant increases in the proportion of omitted medications ( order present but no infusion ) and medications infusing at the wrong dose ( p < 0.05 for both ) . similar to the previous study , when discrepancies in medication infusions were examined by type of medication , the greatest frequency of discrepancies occurred within cardiovascular medication group ( 27% , n = 35 of 128 observations ) ( table 1 ) . discrepancies between orders and pump programing for milrinone and epinephrine infusions increased between study periods ( p < 0.01 for both ) . finally , the anticoagulant group , comprised entirely of heparin infusions for the current study , saw a reduction in discrepancy rate from 46% to 9% ( p < 0.01 ) . in a study performed in 2007 , we observed a high frequency of discrepancies between medication and intravenous fluid orders within a cpoe system and programmed settings on infusion pumps at the beside . subsequently , a bidirectional interface between pharmacy and order entry systems was established , and the icu underwent expansion and relocation . contrary to our hypothesis , while the rate of discrepancies decreased for ivf , the overall frequency of discrepancies among medication infusions did not decrease to a level of statistical significance between study periods . while sample size certainly could alter this finding , given the strong push for adoption of healthcare information technologies , we believe these results are still relevant . furthermore , our analysis suggests that the observed decreases in discrepancies are not solely attributable to the technology and that workflow and other factors are partly responsible for the observed changes . we suspect that this change was related to the impact of the bidirectional interface on the workflow for verbal requests for new medications : previously , if a provider requested that a new medication be made urgently , the pharmacist could deliver the medication to the bedside but was unable to reconcile the order . unless the provider immediately entered the order in cpoe , a discrepancy characterized as an unauthorized medication was created . the bidirectional interface allowed the pharmacist to immediately reconcile the verbal order ( an order communicated verbally from a physician to a pharmacist ) . similarly , the decrease in discrepancies for anticoagulants could be explained by the same mechanism as that just described wherein pharmacists utilize the bidirectional interface to reconcile verbal orders . while unauthorized medications decreased , there was an increase in omitted medications as well as wrong dose discrepancies . we suspect that a change in workflow is the reason for the increase in observed omitted medication discrepancies . around the time of picu relocation , anesthesiologists began placing orders for cardiovascular and sedation / analgesia medications they anticipated would be used intraoperatively , such as an epinephrine or fentanyl infusion , the day prior to scheduled surgery . as such , patients in picu awaiting surgery would have orders for these infusions halted at bedside in the cpoe system but no infusion present until they returned from the operating room the following day . we believe this was responsible for many of the omitted medication discrepancies . the rate of discrepancies between orders and pump settings decreased for fluids , due in large part to a decrease in discrepancies within the total parenteral nutrition subgroup . we have not identified a mechanism by which implementation of the bidirectional interface could account for this decrease . rather , a change in workflow on rounds is more likely responsible for this decrease . between study periods environment pharmacy and dietary presence on rounds increased , resulting in greater collaboration among pharmacists , dieticians , and the providers responsible for ordering tpn . previously , a single patient 's tpn may have been reordered more than once on a given day due to modifications in the composition ordered . we suspect that the described change in workflow allowed for consensus on the desired tpn composition , resulting in fewer total tpn orders and therefore fewer opportunities for discrepancy . little is known about the effects of implementation of systems like this bidirectional interface or other types of closed - loop systems . one found a decrease in mean time from order to administration of iv antibiotics after closed - loop implementation in patient care areas such as general medicine and cardiology . however , due to a small number of observations in intermediate and intensive care areas , the study was underpowered to detect a significant difference in these areas that were examined in our study . the second study found a decrease in medication administration errors ( maes ) for non - iv medications following closed - loop implementation but increased pharmacy and medical staff time to perform tasks . however , medications given via iv infusion , the subject of our investigation , were excluded from this study . while these studies support the use of closed - loop systems , like ours they were not designed to show an improvement in patient outcome or a decrease in patient harm . first , as mentioned , the study was not designed to detect harm , and endpoints such as frequency of adverse events or length of hospital stay were not captured . whether the changes in discrepancies were associated with changes in the incidence of harm to patients or with improvement in other measures of quality of care can not be determined . also , as an uncontrolled before and after study , the ability to draw conclusions about the causes of observed changes is limited and part of the analysis is purely subjective . finally , while the sample size allowed for detection of differences in discrepancy rates within certain groups of medications and ivf , it is possible that the other differences could have been detected with greater numbers in some of the subgroups . fewer discrepancies between ivf orders and programmed settings on bedside infusion pumps were observed following implementation of a bidirectional interface between pharmacy and order entry systems in a picu setting . furthermore , subjective analysis of causation suggests that changes in workflow and other factors are likely to have contributed as much to the observed changes as did the bidirectional interface . study design precluded investigation of any potential association between the changes in discrepancies and changes in patient outcomes or harm . demonstrating such an association is essential in the assessment of the value and efficacy of safety technologies .

pancreatic cancer exhibits profound chemoresistance resulting either from pre - existing ( intrinsic ) mechanisms , or from anticancer drug treatment itself ( acquired chemoresistance ) . we present the case of a patient with pancreatic adenocarcinoma metastatic to the liver who experienced clinical , radiographic and tumor marker response to three lines of gemcitabine - based chemotherapy . the regimens included : 8 cycles of gemcitabine and oxaliplatin ( gemox ) , 8 cycles of gemcitabine , docetaxel and capecitabine ( gtx ) and more than 3 cycles of gemcitabine and nab - paclitaxel , with an exceptional response 2 years from the initiation of chemotherapy for metastatic pancreatic cancer . background . the ability of safety technologies to decrease errors , harm , and risk to patients has yet to be demonstrated consistently . objective . to compare discrepancies between medication and intravenous fluid ( ivf ) orders and bedside infusion pump settings within a pediatric intensive care unit ( picu ) before and after implementation of an interface between computerized physician order entry ( cpoe ) and pharmacy systems . methods . within a 72-bed picu , medication and ivf orders in the cpoe system and bedside infusion pump settings were collected . rates of discrepancy were calculated and categorized by type . results were compared to a study conducted prior to interface implementation . expansion of picu also occurred between study periods . results . of 455 observations , discrepancy rate decreased for ivf ( p = 0.01 ) compared to previous study . overall discrepancy rate for medications was unchanged ; however , medications infusing without an order decreased ( p < 0.01 ) , and orders without corresponding infusion increased ( p < 0.05 ) . conclusions . following implementation of an interface between cpoe and pharmacy systems , fewer discrepancies between ivf orders and infusion pump settings were observed . discrepancies for medications did not change , and some types of discrepancies increased . in addition to interface implementation , changes in healthcare delivery and workflow related to icu expansion contributed to observed changes .

renal transplantation is the unique curative option for patients suffering from end - stage renal disease , but to date the evolution of each patient after transplantation can not be predicted . in the past decades , acute graft rejection has decreased dramatically as a result of the introduction of immunosuppressive drugs . however , immunosuppressive drugs carry undesired and severe side effects such as infections , malignancies , and metabolic disorders which may threaten patient 's life . yet , chronic rejection is still the main cause of long - term graft loss [ 2 , 3 ] . the holy grail of organ transplantation is to maintain long - term graft function without immunosuppressive treatment , namely , operational tolerance ( ot ) . however , ot is a rare event in kidney transplanted patients , as only about 0.03% of cases are estimated to be in such state . thus , despite the efforts made in the past , there is still a clear need to find new strategies to achieve long - term tolerance and to investigate the immunological mechanisms that may be implicated in the process of ot . among the actors implicated in the mechanisms of the immune response , b and t lymphocytes are the main characters that lead to graft rejection . in this play , b lymphocytes have a dual key role since they present antigens of the donor to t cells in addition to secreting antibodies that can lead to acute rejection or , later in time , chronic rejection . nevertheless , a sparse b cell subset has been attributed immune regulatory functions which conveys that not all b cells play on the rejection side . although it was first described in 1974 it was not until 2000 that this population was named regulatory b cells ( breg ) . in the last decade , the regulatory role played by breg has been highlighted by many authors in autoimmune diseases such as systemic lupus erythematosus ( sle ) , rheumatoid arthritis , and pathologies that promote antineutrophil cytoplasmic antibodies and also in allograft tolerance in organ transplantation [ 12 , 13 ] . the current general consensus is that breg develop their function mainly via the secretion of il-10 [ 14 , 15 ] . however , a complete phenotype signature , development pathway , or the immunoregulatory properties of breg have not been fully discovered in mice nor in humans , thus granting future research on this cell type . in this review , our aim is to gather the current knowledge about regulatory b cells and their role in kidney transplantation tolerance in humans and to discuss their potential application as cellular therapeutic agent . one of the darkest spots of breg is their phenotype , since for years researchers in the field have tried through multiple approaches to find unique characteristic markers to define them . there is less discussion about their mechanism of action , which is principally accepted to be il-10 , but the lack of knowledge on what triggers its secretion and the fact that other regulatory mechanisms have also been proposed leave this issue , to date , unresolved . as previously occurred in the studies on regulatory t cells , many researchers have prompted to identify a unique set of markers , transcription factors , or mechanism of action that exclusively identify breg in all contexts . in this sense , genetic and surface expression studies have been conducted with partial success to unravel a unique breg signature [ 16 , 17 ] . also , some hypothesis have been formulated on breg development pathways from a common precursor [ 18 , 19 ] , but the results so far are not conclusive . thus , most authors rely on the capacity to produce interleukin- ( il- ) 10 and on the two main phenotypical signatures used to define breg : ( 1 ) transitional b cell phenotype cd19cd24cd38 and ( 2 ) cd19cd5cd1d ( used in both human and mice ) [ 20 , 21 ] . nevertheless , we still face a lack of specific breg markers , and different phenotypes for il-10-producing b cells with regulatory capacity have been proposed through the years . in 2008 , yanaba and colleagues identified an il-10-producing regulatory b cell subset in mice expressing cd1dcd5 which they referred to as b10 cells . a few years later , the same group characterized a similar il-10-producing b cell subset in humans . human b10 cells ' regulatory potential was shown by their capacity to inhibit tumor necrosis factor- ( tnf- ) production by cd4 t helper cells and monocytes . in peripheral blood , b10 cells were found exclusively among cd24cd27 cells , whereas in spleen no difference was observed between il-10-producing and nonproducing b cells regarding their surface markers . when testing the immunomodulatory capacity of the same subset from patients with allergic asthma in vitro , these cells were less capable of secreting il-10 and inducing the secretion of il-10 from cd4 t compared to the same cell population from healthy subjects , suggesting that this population could hold immunomodulatory capability . nevertheless , matsumoto et al . found that cd27cd38 immunoglobulin- ( ig- ) secreting plasmablasts that arise from nave and immature b cells from human blood are the major il-10-producing b cells after in vitro stimulation . yet , the transitional b cell subset cd24cd38 also seems to have regulatory capacity , since after cd40 stimulation they could suppress the differentiation of nave t cells into t helper 1 ( th1 ) and th17 and lead cd4cd25 t cells conversion into regulatory t cells ( treg ) , partially via il-10 [ 9 , 24 ] . due to the disparity of the results showing that different b cell subsets can express immunomodulatory properties , a current emerging view is that breg are not a specific b cell subset but rather a circumstantial b cell phenotype . in this scenario , b cells could acquire a regulatory role when appropriate signals are generated in the environment , as has been already suggested by some authors [ 2527 ] . it would seem reasonable to think that , depending on the type of activated immune cells and cytokines released to the environment , some b cells could shape their response towards the appropriate way to modulate the response of other immune cells . the adaptability of the breg response could explain the different outcomes depending on the disease studied in vivo or the stimulation provided in vitro . breg marker must be reoriented to find the right stimulation for b cells to become stable regulators of the immune response in a given scenario . as mentioned above , il-10 production is perhaps the principal hallmark to define regulatory b cells , describing their immunomodulatory potential and explaining their mechanism of action . il-10 is a regulatory cytokine secreted by almost all innate and adaptive immune cells that plays an essential role in maintaining immune homoeostasis . it binds as a homodimer to its receptor which is a tetramer formed of two ( il-10r1 ) and two ( il-10r2 ) chains . il-10r1 binds to the cytokine while il-10r2 is responsible for the downstream signaling activation through jak1 and signal transducer and activator of transcription 3 ( stat3 ) . il-10 is the only ligand for il-10r1 which in turn is the unique receptor of il-10 , while il-10r2 is shared by several cytokines such as il-20 , il-22 , il-24 , il-26 , il-28 , and il-29 . although little is known about the molecular pathways involved in il-10 secretion in humans , in mice it is mediated by store - operated ca influx from the endoplasmic reticulum , which is further regulated by the calcium sensors stromal interaction molecule ( stim ) 1 and stim2 . among other biological functions , il-10 promotes the downregulation of antigen presentation by macrophages and dendritic cells and suppresses the production of proinflammatory cytokines such as il-1 , interferon ( ifn)- , and tnf- by cd4 t cells , monocytes , and macrophages [ 29 , 31 ] ( figure 1 ) . besides the functional relevance of il-10 expression , il-10-producing cd19cd24cd38 b cells have also been shown to promote the expansion of il-10-producing foxp3 treg and to play a role in inducing their recruitment to the site of inflammation . in addition , human il-10-producing b cells may block the cd28 inducible t cell costimulator ( icos ) costimulatory pathway , thus blocking t cell activation via phosphorylation of src homology region 2 domain - containing phosphatase-1 ( shp1 ) , a downstream molecule of the il-10 receptor intracellular pathway . the key role of il-10 released by b cells has been also proven in multiple sclerosis ( ms ) patients , who have b cells with impaired il-10 production under cd40 stimulation . similarly , in sle patients , b cells fail to produce il-10 in response to cd40 but not to cpg oligodeoxynucleotides ( cpg ) . these results indicate an impaired t cell - dependent breg induction in both autoimmune diseases . not only il-10 but also other regulatory mechanisms like il-35 , granzyme b ( gzmb ) , transforming growth factor- ( tgf- ) , and indoleamine 2,3-dioxygenase ( ido ) have been suggested as important molecules in breg tolerogenic function . figure 1 depicts some of the different breg inducers , mechanisms of action , and functions described in several studies . in one of them , when cd4cd25 t cells were cocultured with cd40-cpg - stimulated b cells from either healthy controls ( hc ) , immunosuppressive - dependent stable graft function ( si ) patients , or ot patients , the proliferation of t cells was inhibited . when il-10 , tgf- , or gzmb were blocked separately , only the anti - gzmb antibody hindered the inhibitory effect on t cell proliferation . however , in a similar experimental set - up where b cells were stimulated with cpg alone , the blockade of tgf- and/or ido activity led to decreased antiproliferative function of breg in coculture with t cells , suggesting different immunosuppression mechanisms depending on the stimulation . somehow these papers entail an il-10 alternative immunosuppressive mechanism of action rather than a characterizing feature of breg . this might hold true for determined breg subsets and strengthens the idea of diverse breg phenotypes depending on the environment . due to their central role as effector cells in the immune response , particularly in acute organ rejection , t lymphocytes have been one of the main targets of immunosuppressive treatments . b lymphocytes also participate in acute rejection by infiltrating allografts and presenting alloantigens to t lymphocytes , promoting the production of ifn- , il-4 , and il-6 among others cytokines . these cells are also capable of differentiating into plasma cells , switching from antigen presenting cells to antibody secretory cells that may target mhc class i and ii molecules of the graft . to hamper this process , an anti - cd20 b cell depleting monoclonal antibody , rituximab , has been introduced as immunosuppressive treatment for transplanted patients . despite the fact that the use of this drug has increased patients ' survival , it fails to induce chronic unresponsiveness to the graft [ 39 , 40 ] . one of the possible reasons underneath may be that plasmablasts and plasma cells , two key players in chronic rejection , do not express cd20 on their cell surface . an additional explanation may be that breg are also depleted by the treatment , thus hampering their tolerogenic function . in this sense , some studies have shown that preserving the b cell compartment favors ot in renal transplantation . seminal papers coled by us and uk consortia ( iot , riset , and itn ) [ 42 , 43 ] showed a similar transitional - breg - related gene signature corresponding to immunosuppressant - free spontaneous ot kidney transplant patients . using microarray analysis and real time pcr , they identified a b cell specific gene signature and different b cell subpopulations distribution in ot patients compared to si patients after transplantation . the signature proposed by newell et al . , relating ot patients to hc but not to their is counterparts , includes 30 genes , most of them are encoding for the / light chains of ig . in the cross - validation experiments , three of these genes were found to be the most predictive : igkv1d-13 , igll1 , and igkv4 - 1 . on the other hand , flow cytometry analyses revealed an increased number of total and nave b cells in ot with respect to si patients . transitional b cells ( defined by the group as cd19cd24cd38igd ) were also found to be increased in tolerant patients and that was consistent in both itn and iot cohorts . since then , several other groups have showed similar traits in their ot or si patients [ 13 , 44 ] . reported that tolerant patients showed a higher frequency of transitional ( defined as cd20cd24cd38 ) and nave ( defined as cd20cd24cd38 ) b cells and a higher production of il-10 compared to si patients . in line with this observation , patients with chronic antibody mediated rejection after renal transplantation were found to have less percentage and absolute numbers of transitional b cells ( defined as cd19cd24cd38 ) when compared to the group of si patients . a recently published update of the itn study revealed a maintained gene signature among ot patients but surprisingly the gene set also increased over time in those si patients . flow cytometric analysis of the b cell population shows a persistent increase in total , nave , and transitional b cell population in ot compared to si patients . furthermore , additional studies have compared transitional / breg frequencies in ot , si , hc , and also chronic rejection patients . interestingly , the last group shows low levels of transitional b cells comparable to the ones of si patients . drawing on the correlation between kidney transplant tolerance and regulatory b cells , the prognostic value of pretransplantation transitional / regulatory b cells and transplantation outcome has been approached [ 48 , 49 ] . in a prospective study , shabir and colleagues show that only higher transitional b cell frequencies before transplantation , but not regulatory t cells , total b cells , or memory b cells , correlate with lower incidence of biopsy proven acute rejection . moreover , patients lacking transitional b cells three months after transplantation are at higher risk of suffering from both t cell and antibody mediated rejection . altogether these studies suggest a marked role of the transitional b cell compartment in graft acceptance and tolerance achievement , which implies that transitional b cells and breg are at least partially overlapping populations . it still remains unclear whether the tolerogenic effect is only created by the breg present in the recipient or whether they can be induced in any patient to generate a tolerance status . since breg and transitional b cells have been acknowledged as a key cell type in the induction and maintenance of tolerance , several groups have studied the effect of different treatments on these b cell compartments in the human setting . the study of the b cell subsets profile in patients under different immunosuppressive regimes has been approached by some groups . the results reported so far have demonstrated that neither mtor nor calcineurin inhibitors ( cni ) induce transitional nor regulatory b cells [ 5052 ] . although mtor inhibitors have shown treg inducing capacity , this effect seems to be breg independent . further , the in vitro study of the effect of cni revealed that it inhibits il-10 expression of b cells . other immunosuppressive agents , such as the b cell depleting antibodies alemtuzumab ( anti - cd52 ) and rituximab ( anti - cd20 ) , have also been tested in transplant patients for their capacity to induce breg . alemtuzumab treated patients show a transient increase in transitional b cells along with a sustained increase in nave b cells . conversely , rituximab has produced far more controversial results . while a single prophylactic dose seemed to protect from developing acute cellular rejection and even induce a b cell repopulation based on transitional b cells , a clinical trial using two doses of the same compound on days 0 and 7 after transplantation had highly deleterious effects , causing excessive rates of acute cell rejection which forced the premature termination of the trial . these studies may suggest that there is a window of time- and dose - dependent effect of b cell depletion to induce regulatory or effector b cell subsets in patients under these treatments . finally , next generation blockers of the b cell function which are being approached in autoimmune diseases , such as belimumab ( b cell activating factor ( baff ) blocker ) or atacicept ( transmembrane activator and calm interactor ( taci ) blocker , affecting both baff and a proliferation - inducing ligand ( april ) ) , will undoubtedly also have an effect on the b cell profile of patients , but to date there is no information on their effect on the breg population . beyond conventional immunosuppressive treatments , mesenchymal stem cells ( msc ) therapy is one of the leading nonpharmacological therapies in transplantation . several clinical trials have approached their tolerogenic potential and a few brought their attention to breg induction . patients with refractory chronic graft versus host disease ( cgvhd ) present lower frequencies of total b cells and cd5il-10 b. however , after three months of msc treatment patients showed improvement of their symptoms correlating with increased cd5il-10 b cells . of note , in another phase ii multicenter clinical trial , lymphocyte subsets were analyzed in patients infused several times with umbilical cord - derived msc to treat cgvhd . although there were no differences between the control and the treated groups regarding b cell numbers , the number of particular cd27 b cells was higher in the treated group after some months of msc infusions , and the clinical symptoms improved . an additional way to induce il-10 and functional breg may rely on helminths infections . individuals infected with schistosoma haematobium have higher percentage of il-10 producing b cells that are able to induce treg and il-10 production by t cells in coculture . moreover , helminthic infection of ms patients has shown therapeutic potential since those patients that were infected presented less clinical symptoms compared to noninfected ms subjects . the authors determined that b cells from helminths - infected ms patients produced more il-10 than noninfected ms patients , and that these il-10 producing cells had a phenotype similar to nave b2 cells . altogether these studies show the potential of several compounds and therapeutic approaches to induce breg . however , the significance of the increase of this particular b cell subset and their specific role in the progression of the disease or the therapeutic effect still need to be fully determined . hence , a proper knowledge on breg is mandatory to monitor the efficacy of the treatment as well as the tolerogenic status of the patient . in view of the potential of breg , many efforts have been made trying to find out how to effectively induce breg in vivo and to deepen into the mechanisms of action underlying breg induction . this vast knowledge is of paramount importance to get more insight into the potential mechanisms and therapeutic targets to induce breg in vivo , strategies for ex vivo induction for forthcoming cell therapy - based approaches , and purification of breg for their further study and characterization . the cell source to purify b cells and to produce breg in vitro differs from one lab to the other . while most groups use peripheral blood mononuclear cells as the main source of b cells due to the easy accessibility of blood , other sources such as lymph nodes ( i.e. , per indication from removed tonsils ) or spleen ( i.e. , discarded organ from cadaveric organ donor ) may also be important to get even larger numbers of b cells . although the levels of expression of some surface markers could vary between b cells from different compartments , little is known about how this can affect the induction of breg in in vitro experiments . cd19 is expressed from the early pro - b cell stage to the b cell lymphoblast stage , but the expression is downregulated upon b cell maturation to plasma cells . aiming at minimizing b cell activation induced by cd19 ligation , many other groups use cd19 negative selection to purify b cells . cd43 is expressed on activated b cells , plasma cells , cd5 b-1a cells , and non - b cells , thus resulting in a good marker to isolate untouched resting mature b cells . alternatively , cd22 is expressed on the surface of mature b cells in peripheral blood , but not on plasma cells or early stages of b cell differentiation , resulting in the isolation of untouched cd19 b cells . as the only current defining characteristic of regulatory b cells is their capacity to secrete il-10 , induction of breg from b cells is usually measured based on the proportion of il-10-producing b cells . although the intracellular pathways are not well known yet , they seem to be inducible in different ways . ligation of cd40 , b cell antigen receptor ( bcr ) , and/or toll - like receptors ( tlr ) together with il-2 or il-4 are the most used stimulating factors . however , a consensus regime to induce il-10 producing breg is still to be defined . decreased expression of tlr9 due to polymorphisms in the tlr9 gene can increase predisposition to sle in humans , which suggests that this is a key factor in breg induction . tlr9 ligation to induce breg can be achieved basically with cpg type b ( generally 2006 ) . lipopolysaccharide ( lps ) , which ligates to tlr4 , is more frequently used in mice since human b cells express very low levels of this receptor in physiological conditions . however , stimulation with anti - igm , cd40l , and il-4 can increase tlr4 expression by human b cells . found that both lps and cpg induced il-10 b cells and cd40 ligation enhanced this effect . also , april has been demonstrated to promote the generation of il-10 producing b cells via stat3 induction . compared to nave b cells from peripheral blood , nave b cells from cord blood seem to have a higher capacity to produce il-10 after stimulation , which could be related to a higher level of pstat3 after cd40 stimulation . this observation reinforces the importance of the source of b cells for ex vivo expansion . our group has recently demonstrated that msc support b cell survival and have a direct effect on their differentiation . when b cells derived from tonsils were stimulated with bcr plus cd40 ligation in the presence of il-2 , plasmablasts were induced . but when b cells in the same setting were cocultured with msc derived from adipose tissue , plasmablast formation was abrogated and breg ( cd19cd24cd38il-10-producing b cells ) were induced . similar results were obtained using b cells from blood , as when they were cultured together , msc promoted the survival and proliferation of b cells and increased the cd5 b cell subset , which has also been described to have immunoregulatory capacity . even though the mechanisms underlying these effects are unknown , in the same study they showed that inhibition of the ido pathway partially reduced the effect of msc on b cells , while blockade of cox-2/prostaglandin - e2 pathway , il-6 , or il-10 did not have any effect . based on the studies summarized in this review , and also in other studies that have not been mentioned due to space limitation , it is clear that breg may be envisaged as an additional approach for promoting tolerance in several pathologic situations . cell therapy is not a new concept anymore and even in the solid organ transplantation field protocols and clinical trials are being set up to promote tolerance in the absence or in a minimized immunosuppressive regime . msc therapy has taken the lead in this area with several trials done and published in kidney , liver , and bone marrow transplantation . in parallel , regulatory immune cell types such as regulatory t cells , tolerogenic dcs , or regulatory macrophages are the main immune cell types being studied and used for cell therapy in human organ transplantation . the one study , a cooperative project that aims at developing immunoregulatory cell therapies for organ transplanted patients , is the paradigm since they compile and share the knowledge among the research groups devoted to that field . however , to this moment , there are no trials on the use of breg as a cell therapy . the incomplete knowledge on breg induction , stability , and functional potential and the lack of a consensus breg signature are just some of the hurdles to be bypassed to generate a safe and efficient cell product . we might be dealing with different subsets of breg depending on the induction cocktail and system used that might present different stability and functionality . since most of the induction systems used at this moment promote activation of b cells , fillatreau and colleagues propose a method to induce il-10 expression on resting b cells to generate tolerogenic b cells which are poor immunogenic and present a lower potential risk of switching into effector b cell . in contrast to dendritic cells , b cells can not phagocyte an antigen to present it on their surfaces but instead it needs to be recognized by specific bcr , internalized , and presented in mhc - ii . it is unknown whether this antigen is inducing specific tolerance , but if this was the case , it would be necessary to find out how to generate antigen - specific breg . the effect that donor or recipient - derived breg could have in modulating the immune reaction remains unknown if we envision a therapy in the field of organ transplantation or the effect of autologous or allogeneic breg in autoimmune diseases . identifying the mechanism of action by which one and not the other could induce allograft tolerance can shed light on the role of direct and indirect pathway of antigen presentation and tolerance induction . moreover , the age of the patient is a relevant factor in the capacity of regulatory b cells to produce il-10 since it is impaired in cd38cd24 b cells from old individuals independently of the stimulating factor used ( cd40l , phorbol 12-myristate 13-acetate ( pma)/ionomycin , or cpg ) . there are other mechanistic issues that would have to be addressed such as the time needed to produce enough breg , infusion timing and dosage , route of administration , and gmp compliance . in addition to all the above mentioned issues , we are facing an added difficulty in the development of such a therapeutic strategy : the model . in mice models , multiple studies show that breg can induce treg and are capable of transferring tolerance in allogeneic cardiac allograft , islet allograft , and arthritis models . these studies point out the central role of il-10 in the modulation of the immune response . also , another study with islet allograft full mch - mismatched model suggested that treg induction by breg could be mediated through tgf- . in a rat cardiac allograft model , transferred breg from tolerant animals migrated to the graft where they maintained their regulatory capacity . back to mice , t cell ig mucin protein-1 ( tim-1 ) also seems to be a key molecule for breg since tim-1 b cell subset is highly enriched for il-10 producing cells , and the secretion of il-10 increases substantially after tim-1 ligation . nevertheless , it is proven that murine and human breg are essentially different and that their ex vivo induction involves different mechanisms and molecular pathways . in this scenario , we will have to rely on human cell culture approaches and humanized mouse models in order to develop a therapeutic strategy . besides the generation of therapeutic protocols to induce breg in vivo and taking into account the achievements in in vitro expansion of breg , one can envision in the near future a cell therapy approach using breg to promote tolerance . regulatory b cells are one of the newest members of the regulatory immune cells family . many researchers in the field of transplantation and autoimmune diseases have turned their attention to this cell type for their implication in maintaining homeostasis and achieving a tolerant state . while patients suffering from autoimmune diseases such as sle or ms have shown nonfunctional breg populations [ 9 , 81 ] in the transplantation field , higher pretransplantation breg numbers have been associated with lower antibody mediated rejection in kidney transplant recipients . further , in kidney transplantation , breg have become highly interesting due to their association with tolerance [ 12 , 42 , 43 ] . however it still remains elusive whether the increase in breg is cause or consequence of the tolerance status . despite the increasing number of papers published about breg , one of the main hurdles in their study is the absence of a breg signature , and the fact that murine breg are substantially different to the human ones is hindering this endeavor . until now the human breg signature has been mainly resolved by the use of transitional b cell phenotype and/or the ability to secrete il-10 but other extracellular markers and released cytokines have been associated with this cell type . the variability of the breg signature might be due to different breg phenotypes depending on the disease , the activation milieu , or the cell origin . unraveling a proper set of markers that identify this regulatory subset will help in the monitoring of patients and will bring new light into their relation with the immune homeostasis . . the generation of a breg in vitro might be the key point to regain the lost tolerance status opening new doors to the development of innovative therapies . epithelial cells can contribute to chronic inflammatory disorders , synthesizing and secreting a variety of proinflammatory cytokines , such as il-8 , which regulates the neutrophil accumulation in the airways of chronic obstructive pulmonary disease ( copd ) subjects [ 1 , 2 ] . in response to proinflammatory stimuli in addition , in human bronchial epithelial cells , the nfb activation may regulate il-8 release via erk / map kinase - dependent or kinase - independent processes . cigarette smoke , source of oxidative stress in the airways , is able to induce mucin synthesis in epithelial cells and a subsequent goblet cell production . the mucociliary dysfunction component of copd is due to mucus hypersecretion coupled with a decrease in mucus transport , and it is an important pathophysiological feature requiring appropriate treatment . il-17a , a major product of th17 cells , is implicated in the pathogenesis of several inflammatory and autoimmune diseases [ 7 , 8 ] . il-17a is involved in the development and progression of inflammatory diseases of the airways , including allergic asthma , rhinitis , and copd [ 911 ] . recent studies have demonstrated the effect of il-17a on il-8 secretion in the airway epithelial cells and in airway smooth muscle cells [ 9 , 12 , 13 ] . furthermore , il-17a promotes the growth of airway epithelial cells through erk - dependent signaling pathway and is able to generate muc5ac by nfb in bronchial epithelial cells . acetylcholine ( ach ) it is synthesized by choline acetyl - transferase ( chat ) in different cell types ( macrophages , t - lymphocytes , fibroblasts , and epithelial cells ) acting as an autocrine / paracrine growth factor in regulating various aspects of the innate mucosal defense mechanisms including mucociliary clearance and regulation of macrophage function . nonneuronal ach is involved in the activation of bronchial epithelial cells and alveolar macrophages as well as in the release of chemotactic mediators for eosinophils and neutrophils . it contributes to the inflammatory processes of copd via the activation of muscarinic receptors ( mrs ) m1 , m2 , and m3 [ 15 , 21 , 22 ] , and it is involved in the th17 immunity of copd patients . cholinergic agonists promote mucociliary clearance and the release of inflammatory mediators from airway epithelial cells . anticholinergic drugs , currently used in the treatment of copd , block the activity of muscarinic receptors in the airway secretory cells and in smooth muscle , reducing vagal tone and mucus secretion in copd [ 25 , 26 ] . furthermore , in vitro , anticholinergic drugs control the release of ltb4 in the inflammatory cells recovered from induced sputum of copd . we investigated whether il-17a , via erk1/2 and nfb signal pathway activation , is able to modulate the synthesis of chat promoting the autocrine ach release and binding on the cell surface of 16-hbe , in an in vitro model of bronchial epithelial cell line . furthermore , we studied whether the autocrine ach activity , induced by il-17a , promotes the production of il-8 and muc5ac in bronchial epithelial cells . finally , we tested the effectiveness of tiotropium bromide ( spiriva ) , anticholinergic drug usually used in the treatment of copd , in our in vitro model of bronchial inflammation . the sv40 large t antigen - transformed 16-hbe cell line , an immortalized normal bronchial epithelial cell line , or primary normal human bronchial epithelial ( n - hbe ) cells ( atcc , catalog number pcs-300 - 010 ) were used in this study . d. gruenert laboratory ( university of california , san francisco , california ) to ibim - cnr , italy . the cells represent a clonal diploid ( 2n = 6 ) cell line isolated from human lung . evidences showed that 16-hbe cells are similar to primary normal human bronchial epithelial ( n - hbe ) cells and to bronchial epithelial cells from bronchial brushings concerning the response to proinflammatory stimuli and anti - inflammatory drugs . 16-hbe cells and n - hbe cells were cultured as adherent monolayers in eagle 's minimum essential medium ( mem ) supplemented with 10% heat - inactivated ( 56c , 30 min ) fetal bovine serum ( fbs ) , 1% mem ( nonessential amino acids , euroclone ) , 2 mm l - glutamine , and gentamicin 250 g / ml at 37c in a humidified 5% co2 atmosphere . viable 16-hbe and n - hbe cells ( 5 10 ) were seeded into six - well plate in mem 10% fbs and cultured until confluence ( 7080% ) . cells were then made quiescent by culturing for 24 h in mem serum - free . the 16-hbe cells were then stimulated with rhil-17a ( 050 ng / ml ) ( r&d systems , minneapolis , mn , usa ) for 30 minutes to test the activation of the intracellular signal pathways and for 24 h to evaluate the intracellular expression of chat ( protein and mrna ) , ach expression , il-8 release , and muc5ac production . the n - hbe cells were stimulated with rhil-17a 20 ng / ml for 24 h to evaluate the intracellular expression of chat ( protein and mrna ) , ach expression , il-8 release , and muc5ac production . we tested the effects of 2-(2-amino-3-methoxyphenyl)-4h-1-benzopyran-4-one ( pd098,059 , an inhibitor of mapkk activation , 25 m ) ( sigma aldrich s.r.l . , milan , italy ) and ( e)-3-(4-methylphenylsulfonyl)-2-propenenitrile ( bay11 - 7082 , an inhibitor of ikb phosphorylation , 50 m ) ( sigma aldrich s.r.l . , milan , italy ) in 16-hbe cells stimulated in the presence and absence of rhil-17a 20 ng / ml . furthermore , we tested the effect of hemicholinium-3 ( hch-3 ) ( a potent and selective choline uptake blocker , 50 m ) ( sigma aldrich , milan , italy ) in 16-hbe cells stimulated in the presence and absence of rhil-17a 20 ng / ml . finally , we studied the effects of anticholinergic compound tiotropium ( spiriva ) ( 100 nm ) ( boehringer ingelheim pharma gmbh & co. kg , biberach , germany ) in 16-hbe and n - hbe cells stimulated in the presence and absence of rhil-17a 20 ng / ml . the drugs were added to the cells medium 1% fbs ( 5% co2 at 37c ) 1 h before the stimulation with rhil-17a . the expression of chat protein was determined in 16-hbe and n - hbe cells treated with rhil-17a for 24 h or medium alone using indirect label immunofluorescence analyzed by flow cytometry ( facstar plus analyzer , becton dickinson , mountain view , ca ) as previously described . furthermore , the expression of chat protein was measured by western blot analysis as previously described . the primary mouse anti - chat antibody ( mab5270 , chemicon , millipore ) was used for both flow cytometry and western blot . total rna was extracted from 16-hbe cells with trizol reagent ( invitrogen ) following the manufacturer 's instructions and was reverse - transcribed into cdna , using m - mlv - rt and oligo(dt)1218 primer ( invitrogen ) . quantitative real - time pcr of chat transcripts was carried out on steponeplus real - time pcr system ( applied biosystems , foster city , ca , usa ) using specific fam - labeled probe and primers ( prevalidated taqman gene expression assay for chat , hs00253141 m1 , assays - on - demand , applied biosystems ) . the expression of chat gene was normalized to glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) endogenous control gene . relative quantitation of gene expression was carried out with the comparative ct method ( 2 ) and it was plotted as fold change compared with untreated cells as the reference sample . the 16-hbe cells were collected , washed in cold pbs , and incubated 1 h at 4c with a rabbit polyclonal anti - ach antibody ( ab8884 , abcam , cambridge , uk ) . after washing with cold pbs , fitc - conjugated polyclonal swine anti - rabbit igg ( dako , glostrup , denmark ) was added to the cells for 30 min at 4c . fluorescence - positive cells were analyzed using facscalibur flow cytometer ( becton dickinson , mountain view , ca , usa ) . the percentage of positive cells was determined from forward scatter ( fs ) and sideways scatter ( ss ) patterns . no specific binding as well as background fluorescence was detected by analyzing negative control samples . it was measured in protein extracts from cultured 16-hbe cells by a fluorimetric method using a commercial kit ( biovision research products , ca , usa , cat . # the kit detects choline ( ch ) and total choline ( tch ) by adding acetylcholine esterase to the reaction that converts ach into ch with sensitivity until 50 pmol / well by plotting fluorescence readings ( ex / em 535/587 nm ) against the standard curve . this sensitivity is correspondent to the concentration of 1 m of tch or ch . fluorescence intensity was read using a wallac 1420 victor multilabel counter ( perkinelmer life sciences , turku , finland ) . the activation of erk1/2 and nfb pathway was performed by western blot analyses as previously described . we used two rabbit monoclonal antibodies against anti - phospho - erk1/2 and against anti - phospho - ikb ( cell signaling technology , beverly , ma ) , respectively , and an anti--actin antibody ( sigma , st . the cells were washed with cold pbs and nuclear / cytoplasmic extracts were obtained using an ne - per nuclear and cytoplasmic extraction kit ( thermo scientific , rockford , usa ) , which ensures efficient cell lysis and extraction of separate cytoplasmic and nuclear protein fractions by centrifugation . then , 2530 g of the lysate was denatured under reducing conditions by boiling for 3 min in 50 mm tris - hcl ( ph 6.8 ) , 1% sodium dodecyl sulfate ( sds ) , 2% -mercaptoethanol , and 0.01% bromophenol blue . the nuclear and cytoplasmic proteins extracts were separated by sds - polyacrylamide gel electrophoresis ( page ) and transferred via electrophoresis onto immobilon - p membranes ( millipore , bedford , ma , usa ) . after transfer , the membranes were blocked overnight at room temperature in pbs containing 3% bsa and 0.5% tween 20 and then incubated for 1 h at room temperature with anti - nfb - p65 ( c-20 ) sc-372 , santa cruz biotechnology ( santa cruz , ca , usa ) . 16-hbe and n - hbe cells were previously fixed with 4% paraformaldehyde in pbs , washed , and permeabilized with pbs ( containing 0.1% saponin , 1% sodium azide , and 10% fbs ) and incubated with primary antibody , mouse monoclonal anti - muc5ac clone 45m1 ( 200 g / ml , # ms-145-p , neomarkers , fremont , ca ) . the cells were washed twice and then incubated with fitc - conjugated rabbit polyclonal anti - mouse igg f(ab)2 ( dako , glostrup , denmark ) for 30 min at 4c and then analyzed by flow cytometry analyses ( facscalibur flow cytometer , becton dickinson , mountain view , ca , usa ) supported by cellquest acquisition and data analysis software ( becton dickinson , mountain view , ca , usa ) . the cells were considered muc5ac positive when their fluorescence 1 ( fl1 ) was greater than a gate set to exclude all cells in the fl1 peak from an isotype matched control antibody . 16-hbe and n - hbe cells were lysed with lysis buffer ( 10 mm tris - hcl ( ph 7.4 ) , 50 mm nacl , 5 mm edta , and 1% np-40 nonidet ) containing protease and phosphatases inhibitors and frozen at 20c . after being defrozen , samples were centrifuged at 12000 rpm for 20 min , supernatants were recovered , and the protein concentration was quantified by bradford solution . equal amounts of cell extract proteins ( 40 g / lane ) were separated via sds - page electrophoresis in 8% acrylamide - bisacrylamide ( 80 : 1 ) under nonreducing conditions [ 32 , 33 ] . the proteins were then transferred electrophoretically to nitrocellulose membranes that were incubated with 5% fat - free skimmed milk in pbs for 1 h and then incubated with the same antibody and then western blot ( dilution 1 : 500 , overnight at 4c ) . subsequently , the membrane was washed and incubated for 1 h with a secondary antibody conjugated to hrp . after wash in pbs 1x , detection was performed with an enhanced chemiluminescence system ( ambion , austin , tx ) followed by autoradiography . the levels of il-8 were determined in 16-hbe supernatants using a commercial elisa kit ( r&d systems , inc . furthermore , the levels of il-8 expression were determined in 16-hbe and n - hbe protein extracts by western blot using a mouse monoclonal anti - il-8 ( dilution 1 : 100 , overnight at 4c ) ( sc-8427 , santa cruz biotechnology , inc . ) and a sheep anti - mouse igg - hrp ( dilution 1 : 1000 ) ( na931 , ge healthcare uk , little chalfont , buckinghamshire , uk ) as primary and secondary antibody , respectively . cell transfection was performed according to the manufacturer 's instructions ( 72 h at 37c ) in siport neofx transfection agent ( ambion , inc . , texas , usa ) diluted in opti - mem ( gibco invitrogen , milan , italy ) and chat sirna ( 20 m , s-2969 ambion , inc . , texas , usa ) diluted in opti - mem for a final concentration of 30 nm . a sirna ( 20 m , ambion , inc . , texas , usa ) , containing a scrambled sequence without significant homology to the human genome , was used as negative control . furthermore , the effect of chat sirna was checked in il-8 measurement and in muc5ac production obtained as mentioned above . gel images were taken with epson gt-6000 scanner and then were imported into national institutes of health image analysis 1.61 program to determine band intensities . data are expressed as arbitrary densitometric units ( adu ) corrected against the density of -actin bands . analysis of variance ( anova ) corrected with fisher 's test and t - test was used for comparisons . the stimulation of 16-hbe cells with rhil-17a ( 20 and 50 ng / ml for 24 h ) significantly increased chat protein expression compared with unstimulated cells by both flow cytometry ( figures 1(a ) and 1(b ) ) and western blot analyses ( figure 1(c ) ) . the stimulation of the cells with rhil-17a 20 ng / ml reached the higher levels of chat synthesis ( figure 1 ) . accordingly , we showed that the levels of chat mrna , obtained by rt - pcr , significantly increased in 16-hbe cells stimulated with rhil-17a 20 ng / ml compared with unstimulated cells ( figure 1(d ) ) . finally , we showed that rhil-17a ( 20 and 50 ng / ml for 24 h ) significantly increased the ach binding ( figures 2(a ) and 2(b ) ) and production ( figure 2(c ) ) compared with unstimulated cells . the stimulation of the cells with rhil-17a 20 ng / ml reached the higher levels of ach binding and production ( figure 2 ) . the pikb and perk1/2 were significantly increased in 16-hbe cells stimulated with rhil-17a 20 ng / ml for 30 min in comparison with untreated cells , reaching higher levels when compared with the cells stimulated for 2 h with rhil-17a 20 ng / ml ( figures 3(a ) and 3(b ) ) . the dose - response curve showed a significant increase of pikb and perk1/2 in 16-hbe cells stimulated with rhil-17a 20 ng / ml in comparison with unstimulated cells . the levels of pikb and perk1/2 shaped in 16-hbe cells stimulated with rhil-17a 50 ng / ml in comparison with the cells stimulated with rhil-17a 20 ng / ml are shown in figures 3(c ) and 3(d ) . finally , we showed an increase of nfb - p65 nuclear translocation in 16-hbe cells stimulated with rhil-17a 20 ng / ml compared with untreated cells ( figure 3(e ) ) . the preincubation of 16-hbe cells with pd098,059 or bay11 - 7082 significantly reduced the expression of chat protein in 16-hbe cells stimulated with rhil-17a 20 ng / ml for 24 h by both flow cytometry ( figures 4(a ) and 4(b ) ) and western blot analysis ( figure 4(c ) ) . accordingly , the pretreatment of the cells with pd098,059 and bay11 - 7082 significantly decreased ach binding ( figures 5(a ) and 5(b ) ) and production ( figure 5(c ) ) in 16-hbe cells stimulated with rhil-17a 20 ng / ml compared with the cells stimulated with rhil-17a alone . finally , the use of tiotropium significantly decreased the ach binding in 16-hbe cells stimulated with rhil-17a 20 ng / ml compared with the cells stimulated with rhil-17a alone ( figures 5(d ) and 5(e ) ) . the stimulation of 16-hbe cells with rhil-17a 20 ng / ml for 24 h significantly increased the levels of il-8 protein expression and release compared to unstimulated cells ( figure 6 ) . the pretreatment of the cells with hch-3 ( 50 m ) or tiotropium ( 100 nm ) significantly reduced the levels of il-8 protein expression ( figures 6(a ) and 6(b ) ) and release ( figure 6(c ) ) in 16-hbe cells stimulated with rhil-17a 20 ng / ml compared with the cells stimulated with rhil-17a alone . additionally , the stimulation of 16-hbe cells with rhil-17a 20 ng / ml for 24 h significantly increased muc5ac expression , compared with unstimulated cells evaluated by both flow cytometry and western blot analysis ( figure 7 ) . the pretreatment of the cells with hch-3 ( 50 m ) or tiotropium ( 100 nm ) significantly reduced the muc5ac protein expression in 16-hbe cells stimulated with rhil-17a 20 ng / ml compared with the cells stimulated with rhil-17a alone ( figure 7 ) . the temporary transfection of 16-hbe cells with chat sirna generated a significant decrease in muc5ac expression ( figures 8(a ) and 8(b ) ) and il-8 release ( figure 8(c ) ) in 16-hbe cells stimulated with rhil-17a 20 ng / ml , when compared with unsilenced or scrambled cells stimulated with rhil-17a . knockdown efficiency of chat mrna ( 40 2.5% ) was obtained comparing the levels of chat protein expression in silenced and scrambled unstimulated 16-hbe cells ( figure 8(d ) ) . the stimulation of n - hbe cells with rhil-17a 20 ng / ml for 24 h significantly increased the levels of chat , muc5ac , and il-8 protein expression compared with unstimulated cells . the pretreatment with tiotropium ( 100 nm ) significantly reduced the levels of muc5ac and il-8 protein expression in n - hbe cells stimulated with rhil-17a 20 ng / ml ( figure 9 ) . in our in vitro model , we demonstrated for the first time that il-17a , via erk1/2 and nfb pathway activation , is able to generate an increased synthesis of chat protein promoting ach production and binding in human bronchial epithelial cells . the autocrine ach activity observed in the presence of il-17a increased the levels of il-8 and muc5ac production in human bronchial epithelial cells . finally , anticholinergic drugs such as tiotropium ( spiriva ) might have anti - inflammatory and antisecretory properties by the control of ach activity generated in bronchial epithelial cells stimulated with il-17a . much of our knowledge comes from the interactions between environmental and inflammatory stimuli , and the airway epithelium has been derived extensively from in vitro cell culture models using transformed 16-hbe cells , representing an invaluable model in understanding the physiological properties of human airway epithelium [ 22 , 27 , 34 ] . in accordance with this concept , our study is based principally on the use of an immortalized airway epithelial cell line , 16-hbe . nevertheless , to strengthen the message of our findings we performed some experiments using commercially available primary bronchial epithelial cells . ach , classically known as a parasympathetic neurotransmitter , is able to affect the inflammatory processes in copd [ 19 , 22 , 35 ] . th17 cells producing il-17a are associated with nonneuronal ach in the systemic inflammation of copd patients . the stimulation of cholinergic receptors enhances il-10 and il-17a and inhibits ifn- secretion in murine spleen t - cells suggesting a link between t - cell , cholinergic receptors , and th17 lineages . furthermore , it was observed that il-17a is a proinflammatory cytokine involved in modulating airway immune response in several aspects covering both the innate and the adaptive immunity in chronic lung disorders [ 3739 ] . bronchial epithelial cells from copd patients as well as 16-hbe cell line express il-17 receptor . in light of this background , we hypothesized a link between il-17a and the production of autocrine growth factor ach in bronchial epithelial cells . accordingly , we found that rhil-17a is able to increase the basal levels of chat protein ( the enzyme involved in the synthesis of ach ) synthesis , the levels of autocrine ach , and the related binding to cellular surface of 16-hbe cells . rhil-17a affects the synthesis of chat protein also in our in vitro experiments with primary epithelial cells from normal donors ( n - hbe ) . these findings might suggest that th17 immunity is involved in the chat / ach activity promoting the bronchial epithelial cells activation during inflammatory process of the airways . a relevant number of studies identified that il-17a promotes the activation of bronchial epithelial cells through erk1/2 or nfb dependent signaling pathway [ 14 , 15 ] . the activation of mek / erk cascade regulates chat promoter via nfb signal pathway in transfected chp126 neuroepithelioma cells . accordingly , we observed higher levels of erk1/2 and ikb phosphorylation in 16-hbe cells stimulated with rhil-17a 20 ng / ml , suggesting the involvement of mek / erk and nfb pathway activation in our in vitro model . furthermore , looking at nfb - p65 in nuclear extract ( as upstream of pikb ) , we underlined an increase of the nuclear translocation of nfb in 16-hbe cells stimulated with il-17a 20 ng / ml . these findings might provide evidences for the involvement of nfb pathway in bronchial epithelial cells stimulated with il-17a . to demonstrate that rhil-17a regulates chat synthesis via mek / erk cascade and via nfb pathway , we preincubated with pd098,059 ( inhibitor of mapkk activation ) and bay11 - 7082 ( inhibitor of ikb phosphorylation ) the 16-hbe cells stimulated with rhil-17a . the use of the specific inhibitors reduced the levels of chat protein synthesis and ach production and binding in the cells stimulated with rhil-17a suggesting the involvement of mek / erk cascade and via nfb pathway in this mechanism of synthesis . however , we observed the dose - response curve of rhil-17a peaks at 20 ng / ml for erk1/2 and ikb signaling to shape at the concentration of il-17a 50 ng / ml . in contrast to these data , we showed persistence of chat synthesis and ach production and binding . these findings might suggest that the higher concentration of rhil-17a might be involved in the chat synthesis , independently of the mek / erk cascade and nfb pathway , by the activation of other intracellular signal pathways . however , in this study we focused our attention on the effect of rhil-17a 20 ng / ml . nevertheless , we suggest that further studies will be necessary to better clarify the role of intracellular signal pathway involved in chat synthesis and autocrine ach activity of bronchial epithelial cells stimulated with rhil-17a 50 ng / ml . th17 immunity has an emerging role in the induction of neutrophilic airway inflammation [ 9 , 12 , 13 ] and muc5ac production . the reduced sensitivity to gcs related to th17 immunity has been clinically associated with neutrophilic airway inflammation [ 42 , 43 ] , but it is still largely unclear which cellular and molecular mechanisms contribute to this phenomenon . il-17a activated the p38 , extracellular signal - related kinase ( erk ) , and phosphoinositide-3-kinase ( pi3k ) pathways inducing gc insensitivity . furthermore , mechanisms whereby pharmacological agents such as glucocorticoids repress mucin gene expression are not well studied , although they might be important for formulating therapeutic interventions in chronic lung diseases . preclinical studies have demonstrated that stimulation with cholinergic agonists ( ach , methacholine ) or cholinergic antagonists ( atropine ) , targeting muscarinic receptors , increases mucociliary clearance [ 24 , 45 , 46 ] . accordingly , anticholinergic drugs block muscarinic receptors activity on airway secretory cells and smooth muscle and so , theoretically , may reduce vagal tone and mucus secretion in copd facilitating cough - induced mucus clearance [ 25 , 26 ] . consistent with this suggestion is the observation that tiotropium reduces levels of mucus secretion in patients with copd . furthermore , tiotropium reduces exacerbation frequency in copd , but this effect does not appear to be due to a reduction in il-8 systemic inflammation and is able to control il-8 release in epithelial cells in vitro [ 22 , 49 ] . in this scenario , we speculate that the blockade of il-17a downstream , regarding chat synthesis , and autocrine ach production might represent a new strategy for therapeutic intervention in gc insensitivity of airway inflammation using anticholinergic drugs . accordingly , we showed that the use of tiotropium is able to reduce autocrine ach binding . these results suggest the involvement of machrs in the activation of bronchial epithelial cells stimulated with il-17a . furthermore , we observed that the use of muscarinic receptor antagonists such as tiotropium is able to control the muc5ac and il-8 production blocking the activity of ach bound to machrs generated by il-17a in bronchial epithelial cells . we support this observation showing the effect of hch-3 ( choline uptake blocker ) that , blocking the synthesis of autocrine ach , reduced the muc5ac and il-8 production in 16-hbe cells stimulated with rhil-17a . finally , we observed that the chat enzyme rna interference promoted the reduction of muc5ac and il-8 production in the 16-hbe cells stimulated with rhil-17a rather than in both unsilenced and scrambled cells stimulated with rhil-17a . these results might suggest the potential involvement of chat / ach pathway in the regulation of mucin production and inflammation in bronchial epithelial cells during chronic airway disease . however , rhil-17a did not affect the expression of machrs ( m13 ) in 16-hbe cells ( data not shown ) . in this scenario , our in vitro model suggests the potential relevant contribution of anticholinergic drugs to the blockade of mucus and inflammatory secretion during copd exacerbations . accordingly , we showed that tiotropium reduced muc5ac and il-8 production in the experiment performed with 16-hbe and primary n - hbe cells stimulated with rhil-17a . nevertheless , further in vivo clinical study should be necessary to clarify the role of anticholinergic drugs in the control of mucus and inflammatory mediator secretion from bronchial epithelial cells during il-17a mediated airway inflammation . to our knowledge , this is the first descriptive study demonstrating the involvement of il-17a in the induction of the chat / ach activity in bronchial epithelial cells promoting inflammation and mucus secretion in the airways . the ability of muscarinic receptor antagonists , including tiotropium , to modulate il-17a activity might open up perspectives for novel therapeutic strategies in the control of th17 immunity during the airway inflammation and the mucus secretion of copd patients ( figure 10 ) .

regulatory b cells ( breg ) are in the spotlight for their role in immune homeostasis maintenance and tolerance achievement as in the last years the correlation with functional and increased breg numbers in autoimmune diseases and transplantation has been extensively proven . their study is , however , in its infancy with still little knowledge and consensus on their origin , phenotype , and mechanism of action . all this hampers the pursuit of an effective breg induction method for therapeutic purposes . in this review we aim to summarize the studies on human breg and their implication in kidney transplantation and to further discuss the issues surrounding therapeutic applications of this cell subset . il-17a is overexpressed in the lung during acute neutrophilic inflammation . acetylcholine ( ach ) increases il-8 and muc5ac production in airway epithelial cells . we aimed to characterize the involvement of nonneuronal components of cholinergic system on il-8 and muc5ac production in bronchial epithelial cells stimulated with il-17a . bronchial epithelial cells were stimulated with recombinant human il-17a ( rhil-17a ) to evaluate the chat expression , the ach binding and production , the il-8 release , and the muc5ac production . furthermore , the effectiveness of pd098,059 ( inhibitor of mapkk activation ) , bay11 - 7082 ( inhibitor of ikb phosphorylation ) , hemicholinium-3 ( hch-3 ) ( choline uptake blocker ) , and tiotropium bromide ( spiriva ) ( anticholinergic drug ) was tested in our in vitro model . we showed that rhil-17a increased the expression of chat , the levels of ach binding and production , and the il-8 and muc5ac production in stimulated bronchial epithelial cells compared with untreated cells . the pretreatment of the cells with pd098,059 and bay11 - 7082 decreased the chat expression and the ach production / binding , while hch-3 and tiotropium decreased the il-8 and muc5ac synthesis in bronchial epithelial cells stimulated with rhil-17a . il-17a is involved in the il-8 and muc5ac production promoting , via nfb and erk1/2 pathway activation , the synthesis of chat , and the related activity of autocrine ach in bronchial epithelial cells .

an estimated 630,000 people worldwide were diagnosed with head and neck squamous cell carcinoma ( hnscc ) in 2008 , representing 6% of all malignancies and making it the 6th most common cancer [ 1 , 2 ] . in the united states , approximately 48,000 cases occurred in 2009 , with 11,300 deaths . however , two - thirds of patients are diagnosed at a locoregional advanced stage ( iii - ivb ) , with 5-year survival rates of 30 to 60% for these patients . in the past decade , treatment of locoregional advanced hnscc has shifted from primary surgery to organ preservation with combination chemoradiotherapy ( crt ) . the current approach attempts to achieve both organ preservation and function with outcomes superior to radiotherapy alone or surgery with postoperative radiotherapy [ 612 ] . a recent meta - analysis by pignon et al . showed an absolute survival benefit of 6.5% at five years when chemotherapy was administered concomitantly with radiotherapy . the mechanism for the survival benefit of crt is thought to occur via increased tumor sensitization to the cytotoxic effects of radiotherapy , while providing adjuvant treatment for potential distant metastatic disease . however , crt has acute and long - term toxicities that can both limit treatment and increase morbidity . furthermore , although several crt regimens produce high rates of complete response at the primary site and regional neck nodes , there exists a high rate of failure to systemically eradicate micrometastases . this is demonstrated by the later occurrence of distant metastases , which account for many cancer - specific deaths . therefore , despite the successful use of numerous crt regimens and the development of multidisciplinary management , the prognosis of locoregional advanced hnscc remains poor . in this study , we review the outcomes of a heterogeneous cohort of patients with locoregional advanced hnscc . all patients were treated with curative intent with a uniform crt regimen that consisted of hyperfractionated radiotherapy and concurrent cisplatin/5-fluorouracil ( 5-fu ) . outcomes evaluated were overall survival ( os ) , locoregional control ( lrc ) , and disease - free survival ( dfs ) . in an irb - approved study design ( greater baltimore medical center irb number 07 - 044 - 11 ) , the medical records of 105 patients with previously untreated , locoregional advanced , nonmetastatic stage iii - ivb squamous cell carcinoma of the oropharynx , hypopharynx , or larynx were reviewed . patients with cancers of the salivary glands , sinuses , or unknown primary sites were excluded , as were patients with recurrent tumors or previous chemotherapy or radiation to the head or neck . patients were evaluated by a head and neck surgeon , medical oncologist , radiation oncologist , dentist , speech pathologist , nurse , and social worker . all cases were discussed prior to therapy and on a regular basis thereafter at multidisciplinary conference . all patients underwent comprehensive head and neck exam , including laryngoscopy and triple endoscopy when necessary , and imaging assessment with chest x - ray , ct , pet / ct , or mri of the head and neck when appropriate . all patients had a histologic diagnosis of squamous cell carcinoma and were staged according to american joint committee on cancer ( ajcc ) guidelines . when possible , tumor samples were tested for hpv-16 dna via in situ hybridization - catalyzed signal amplification as previously described . patient demographic and clinical characteristics collected at baseline included age , gender , race , patient - reported weight loss , and karnofsky performance status ( kps ) . in addition , history of tobacco and alcohol use was collected and categorized as follows : tobacco as nonsmoker , < 20 pack - years ( py ) , 2040 py , 4060 py , or > 60 py ; alcohol as nondrinker , < 7 drinks / week ( social ) , 714 drinks / week ( moderate ) , or > 14 drinks / week ( heavy ) . chemotherapy consisted of cisplatin ( 12 mg / m ) administered over one hour and 5-fu ( 600 mg / m ) over twenty hours , dosed for five consecutive days in an inpatient setting during weeks one and six of radiation therapy . radiation therapy consisted of hyperfractionated doses of 125 cgy delivered twice daily for 2833 days for a total dose of 7075 gy to the primary tumor site , 60 gy to involved lymph nodes , and 50 gy to uninvolved cervical and supraclavicular lymph nodes . patients were treated with 6 mv photon beams , opposed lateral fields with focus blocks , and neck nodes were boosted with electron beam after a cumulative dose of 40 gy . treatment interruptions were minimized as much as possible , and a planned treatment break of one week was included after a cumulative dose of 40 gy . mucositis severity , scored according to the world health organization ( who ) oral toxicity scale , was collected from records of patient visits to speech language pathology and clinical notes of the radiation and medical oncologists . duration of gastrostomy tube dependence was calculated from the initiation of crt until the date of removal . all but seven patients with clinical n2 or n3 disease at the time of presentation underwent planned neck dissection . after treatment , patients returned for followup every 2 - 3 months in years 1 - 2 , every 36 months in years 35 , and every 612 months thereafter , or sooner in the event of a clinical concern requiring closer scrutiny . event time distributions were estimated with the method of kaplan and meier and compared using the log - rank statistic or the proportional hazards regression model . the simultaneous effect of two or more factors was studied using the multivariate proportional hazards model , which was reported with 95% confidence intervals ( cis ) . os was calculated from the date of treatment initiation to the day of death or last followup . dfs was calculated from the date of treatment initiation to the day of recurrence or death , whichever came earlier . lrc was similarly calculated from the date of treatment initiation to the day of local or regional recurrence , whichever came first . factors evaluated for prognostic value included age , gender , race , tumor site , histologic grade , ajcc stage , tumor stage , nodal stage , self - reported weight loss , kps , pretreatment hemoglobin , tobacco and alcohol history , and hpv status . in proportional hazards regression models , variables were entered as categorical effects and the hazard ratios for these factors reflect either its presence or absence . the median followup of surviving patients was 57.6 months ( range 4.6 to 118.8 ) . median survival was 99 months , with 3- and 5-year overall survival rates of 77 and 63% for stage iii and 72 and 58% for stage iv . including both stages iii and iv , the 3- and 5-year os was 75 and 60% . uni- and multivariate analyses of factors associated with survival are listed in figure 1(b ) . on uni- and multivariate analysis , age greater than 55 , primary hypopharyngeal cancers , and advanced t3/t4 tumors were associated with decreased survival , while male gender was associated with increased survival . on univariate analysis only , any smoking history and greater than 40 pack - year smoking history were associated with decreased survival . hpv - positive cancers of the oropharynx were associated with slightly decreased mortality ; however , this was not statistically significant ( p = 0.3 ) . the distribution of deaths from primary head and neck cancer and other causes is shown in table 2 . the average time to locoregional recurrence was 13.8 months ( range 6.3 to 33.9 ) after initiation of crt . the 3- and 5-year rates of lrc were 76 and 68% , respectively ( figure 2(a ) ) . of the five patients who developed regional recurrences , three had undergone planned neck dissection following completion of crt . two of these three patients had viable carcinoma identified in the ipsilateral cervical lymph nodes , while the other patient had no evidence of viable tumor identified in any lymph nodes . the most common site of distant metastatic disease was the lungs ( n = 12 ) . the average time to diagnosis of distant metastasis was 14.1 months ( range 3.2 to 31.9 ) after initiation of crt . the average time to development of any recurrence or metastasis was 14.1 months ( range 3.2 to 33.9 ) after initiation of crt . median disease - free survival was 96 months , with 3- and 5-year rates of 63 and 56% , respectively ( figure 3(a ) ) . uni- and multivariate analyses of factors associated with dfs are listed in figure 3(b ) . hypopharyngeal cancer and t3/t4 tumors were both significantly associated with decreased disease - free survival . eighteen second primary malignancies ( spms ) were diagnosed in 17 patients after the completion of treatment . the most common spm was lung ( n = 7 ) , followed by colorectal ( n = 3 ) , hnscc ( n = 2 ) , prostate ( n = 2 ) , medullary thyroid carcinoma ( n = 1 ) , chronic lymphocytic leukemia ( n = 1 ) , multiple myeloma ( n = 1 ) , and cutaneous squamous cell carcinoma ( n = 1 ) . the average time to diagnosis of spm was 33 months ( range 3.0 to 63.2 ) . twenty - four patients ( 36% ) had grade 3 mucositis , and 39 patients ( 59% ) had grade 4 mucositis . the median duration of peg use was 134 days ( range 311570 ) , which included patients who died with a peg tube in place . thirty - two of 94 patients ( 34% ) required a peg for greater than 6 months , and 10/94 patients ( 11% ) required a peg for greater than 12 months . thirteen out of these 67 patients ( 19% ) developed a stricture , as reported previously . seventy - one of 105 patients were nodal stage n2 or n3 , and 64 patients underwent neck dissection ( 61% ) . of the seven patients with n2 or n3 disease who did not undergo neck dissection , two died prior to surgery , one was too ill to undergo surgery , one refused surgery , and reasons were not available for the other three patients . this rate of viable carcinoma detected post - crt is comparable to that in the cohort of patients with oropharyngeal cancer previously reported by hillel et al . and the use of combination chemotherapy and radiation therapy as primary treatment for locoregional advanced hnscc in medically fit patients has been well established as a treatment option in numerous trials and meta - analyses . furthermore , altered fractionation radiotherapy with concomitant chemotherapy is a well - established treatment for locoregional advanced , nonmetastatic disease . the landmark study by brizel et al . showed that hyperfractionated radiotherapy with cisplatin and 5-fu resulted in improved survival compared to radiation therapy alone . therefore , we sought to review the efficacy of a uniform treatment regimen in a heterogeneous cohort with locoregional advanced hnscc . the results of the concurrent administration of crt for 105 consecutive patients revealed favorable outcomes . for example , two other studies reported 5-year overall survival rates of 59% and 40% , respectively , in cohorts of patients with stage iii or iv disease who received similar radiation and the same chemotherapeutic agents [ 21 , 22 ] . in another series of stage iv patients who received concurrent crt , the 5-year os was 46% . likewise , in the study by brizel et al . , which employed similar hyperfractionated radiotherapy and concurrent cisplatin and 5-fluorouracil in the same doses as our regimen , the 3-year os was 55% and the lrc was 70% . in our cohort , 12 patients ( 11% ) developed locoregional recurrence , and 16 patients ( 15% ) developed distant metastasis . this is comparable to other studies with similar crt regimens , which have reported locoregional recurrence in 2231% of patients and distant metastasis in 1320% of patients [ 21 , 22 ] . the patterns of failure were approximately equal between locoregional ( n = 12 ) and distant sites ( n = 16 ) , which contrasts with several studies in which the majority of failures occur either at the primary tumor site or cervical lymph nodes [ 6 , 12 , 23 ] . reported the results of their experience with two main treatment regimens : type 1 , intensive induction chemotherapy followed by split - course chemotherapy , or type 2 , intensive , split - course hyperfractionated chemotherapy . they reported a 5-year lrc rate of 31 and 17% for types 1 and 2 , respectively , and a 5-year distant failure rate of 13 and 22% for types 1 and 2 . interestingly , the patients who received induction crt were more likely to have locoregional control but were also more likely to have distant metastatic relapse . this has prompted some to suggest the role of induction chemotherapy in treating micrometastatic disease , as other trials have shown a reduction in rates of distant metastasis with the use of induction crt . in our cohort , there were 14 patients who presented with a primary cancer of the hypopharynx and one patient with cancers of the hypopharynx and oropharynx . eleven of the 14 patients ( 79% ) with hypopharyngeal cancer underwent neck dissection , and 6/11 ( 55% ) had viable carcinoma ( versus 28% of the overall cohort who underwent neck dissection ) . eight of 14 patients ( 57% ) had a recurrence , compared to 25/105 ( 24% ) in the overall cohort . furthermore , of the eight recurrences in the hypopharynx group , seven of them presented with distant metastases . these data reflect the poor overall survival of those with hypopharynx cancers in this cohort , which is similar to other published studies [ 2528 ] . patients with hypopharyngeal squamous cell carcinoma may therefore need a different therapeutic approach . given the ongoing debate regarding the possibility of induction chemotherapy to better treat micrometastases , this method of crt should be considered in a direct trial against concomitant crt or perhaps a return to primary surgery with postoperative radiotherapy is warranted . the high rate of grade 3 or 4 mucositis observed in this cohort was consistent with other reports [ 29 , 30 ] . with regard to the elective placement of peg tubes , this is supported by the need to maintain or minimize the weight loss associated with crt , which is between 10 to 14% [ 12 , 30 , 31 ] . in one study , elective versus nonelective use of peg was compared , and elective peg resulted in less weight loss and decreased length of hospitalization . the median duration of peg use and the rate of patients that required a peg tube for longer than 6 or 12 months is similar to other studies [ 3335 ] . furthermore , the exact duration reported may be overestimated due to the retrospective nature of this study and the inability to determine exactly when the patients no longer used the peg tube . patients with head and neck cancer have an increased rate of second primary malignancies , estimated at 39% per year [ 2 , 36 ] . these cancers affect the entire aerodigestive tract , are often related to smoking history , and are therefore of great significance in survivors of head and neck cancer . consistent with these studies , the high rate of second primary lung cancer detected in our patient cohort was not unexpected and was ultimately responsible for a significant fraction of patient deaths after they were without evidence of disease of head and neck cancer . in our cohort , 2 of 18 spm were cancers of the head and neck , similar to another cohort of patients treated with crt . the average time to occurrence of spm was 33 months ( 2.75 years ) in our cohort , similar to other publications of 2.8 years [ 30 , 37 ] . in addition , treatment compliance was high , and few patients experienced unplanned treatment breaks or delays due to excessive toxicity . furthermore , long - term followup was available . only two patients were lost to followup immediately after treatment , and all but six remaining patients had records available from a provider at our institution within the previous year . other strengths included the utilization of a uniform crt regimen , and a thorough chart and database review and availability of the medical records of several departments . despite these strengths , although efforts were made to collect toxicity data , this was often unavailable and not systematically recorded in patient notes ; therefore , documentation of toxicity may have been underestimated and therefore may not be representative . the results of this study show that the use of hyperfractionated radiotherapy with concurrent cisplatin and 5-fluorouracil remains an excellent regimen for the primary treatment of locoregional advanced head and neck cancer . however , the prognosis for patients who experience relapse remains poor , and research focused on predicting which patients will experience these events and what treatment modifications , if any , should be made in order to improve the outcomes for these patients is warranted . lastly , the poor outcomes of patients with cancer of the hypopharynx merit consideration of ongoing research and novel therapies to lessen the mortality of this disease . with the rapid economic development , elevated standard of living , dietary shifts , lifestyle alterations , and aging , diabetes mellitus ( dm ) has become an important public health problem worldwide [ 13 ] , which is estimated to be the third most challenging disease threatening public health after malignant tumors and cardiocerebral vascular diseases . it has been estimated that the global number of individuals with diabetes will double from 171 million in 2000 to 366 million in 2030 among adults aged 20 years . data from european countries have indicated that the health care expenditure for patients with diabetes mellitus was significantly higher than for those who were not diagnosed with this disease [ 68 ] . also , in the united states , it has been estimated that approximately 17.5 million people were diagnosed with insulin - dependent diabetes mellitus ( iddm ) or non - insulin - dependent diabetes mellitus ( niddm ) in 2007 , and the total annual cost in higher medical costs and lost productivity was estimated to be $ 174.4 billion , including $ 159.5 billion for the 16.5 million people with niddm and $ 14.9 billion for the 1 million people with iddm . a recent global study indicated that the prevalence of diabetes mellitus was rising rapidly , particularly in developing countries . moreover , more than 60% of the population with diabetes mellitus all worldwide comes from asia , as this remains the world 's most populous region . the number of persons living with diabetes will increase substantially in each asian country over the next few decades . china is the world 's largest developing economy and the most populous country , with one - fifth of the global population . due to the rapid westernization of diet and lifestyle , the total number of people with diabetes in china is projected to increase from 20.8 million in 2000 to 42.3 million in 2030 . diabetes and its complications also result in significant economic burden among individuals , families , and health care systems . according to who estimates , china will lose $ 558 billion of its national income to heart disease , stroke , and diabetes from 2006 to 2015 . therefore , there is an urgent need to reduce the future diabetes burden by providing adequate financial resources and structures of health care delivery in china , particularly within the context of continued rapid urbanization . diabetes can affect many organ systems throughout the body ( e.g. , nervous system , renal system , and eyes ) and can lead to serious complications over time [ 14 , 15 ] . recently , a meta - analysis confirmed that individuals with diabetes mellitus have an approximately 2-fold higher risk of large - vessel disease , such as coronary heart disease ( chd ) and stroke , and nonvascular mortality . thus , control of the growing prevalence of diabetes mellitus has been widely promoted in order to reduce the risk of large - vessel disease . the pharmacological treatment of diabetes mellitus and lifestyle modification have been shown to decrease the incidence of diabetes mellitus . however , it is very important to first determine the prevalence of diabetes mellitus in the general population , the levels of awareness , treatment , and control of diabetes mellitus . the key elements of effective control include an improvement in the awareness of diabetes mellitus among both health professionals and the general population . some studies [ 2123 ] have revealed that more than half of the individuals with diabetes mellitus were unaware of the condition . furthermore , the percentage of patients with diabetes mellitus who were treated and controlled to target levels was substantially low . moreover , knowing factors associated with the conditions should be useful for health services and public health action in terms of management and prevention . an improvement in the awareness , treatment , and control of diabetes is , therefore , essential for the management and reduction of its prevalence . recently , a systematic review on the prevalence of diabetes was published which included 22 cross - sectional studies . however , there was no information provided about the trends in the prevalence of diabetes and the awareness , treatment , and control of the disease . to date , there has been no systematic review or meta - analysis on the level of awareness , treatment , and control of diabetes in mainland china . thus , the purpose of the current paper was to perform a systematic review and meta - analysis of the published literature regarding the prevalence , awareness , treatment , and control of diabetes in mainland china . the objectives of our study were ( i ) to estimate the trends in prevalence , awareness , treatment , and control of diabetes from 1979 to 2012 in mainland china ; and ( ii ) to estimate the gender , location , and age distributions of patients with diabetes . this systematic review and meta - analysis could provide an overview of the epidemiology of diabetes in the past thirty years in china on the level above the provincial . pubmed , embase , chinese biomedical database ( cbm ) , cnki database , chinese wanfang database , and chongqing vip database were searched from the date of establishment up to february 2013 using the search terms chronic disease , metabolic syndrome , diabetes , diabetes mellitus , prevalence , epidemiology , awareness , treatment , control , cross - sectional survey , longitudinal study and china . all included studies were required to meet the following selection criteria : ( i ) cross - sectional or longitudinal studies that provided the prevalence , awareness , treatment , and control of diabetes in mainland china ; ( ii ) based on population samples rather than volunteers ; and ( iii ) the study population being representative of the provincial or national population . exclusion criteria were ( 1 ) reviews , editorials , letters , commentaries , or reports ; ( 2 ) articles repeating data from other articles that were already included ; ( 3 ) self - reporting data ; and ( 4 ) studies based on special populations , that is , physical examination crowds , industry , or occupational groups , ethnicity , or age groups . we contacted the authors of eligible studies to request further or missing information if needed for subsequent analysis . data regarding the first author , year of publication , study location , survey date , age range , sampling method , location ( urban / rural ) , diagnostic criteria , diagnosis method , total sample size , total case size , gender distribution , age distribution , and prevalence , awareness , treatment , and control of the diabetes were extracted wherever available . an awareness of dm was considered a self - reported previous diagnosis of dm by a physician or other healthcare practitioner among participants with dm . treatment of dm was determined as self - reported if taking oral hypoglycemic medications , using insulin , or other nonpharmacological treatments for the management of high glucose levels . the management of fasting plasma glucose ( fpg ) at levels lower than 7.0 rnmol / l ( 126 mg / dl ) in patients of dm in treatment was defined as control of dm . stata software version 11.1 ( stata , college station , tx , usa ) and review manager ( revman ) version 5.1 were used to calculate the pooled prevalence , awareness , treatment , and control of diabetes from all of the eligible studies . a summary of the prevalence , awareness , treatment , and control estimates was obtained using random - effects meta - analysis . we identified 45,947 references through electronic searches of pubmed ( n = 3,541 ) , embase ( n = 4,013 ) , chinese biomedical database ( cbm ) ( n = 9,547 ) , china national infrastructure database ( cnki ) ( n = 8,189 ) , chinese wanfang database ( n = 9,165 ) , and chongqing vip database after excluding duplicates and following examination of titles , abstracts , and full texts , 172 potentially eligible studies were identified . in addition , with a manual search of references , two more studies [ 27 , 28 ] were obtained from the papers . 57 included repetitive data , 54 studies were researched in specific populations , and 7 studies were self - reports of diabetes prevalence . therefore , we obtained 56 studies in total that were suitable for inclusion in our study [ 2123 , 2779 ] . among the included studies , one study contained survey data for two periods , with the data for 2002 - 2003 being duplicated in another study . we summarized the characteristics of the 56 included studies which contained 7 nationwide studies and 49 provincial studies conducted in the general chinese population . among the included studies , there were 56 concerning the prevalence of diabetes , 10 regarding the awareness of diabetes , 8 involving the treatment of diabetes , and 8 regarding the control of diabetes . a total population of 2,058,243 individuals was investigated , and 120,992 patients with diabetes were detected . the 56 studies were conducted among all of the provinces of mainland china , and included 22 provinces , 4 municipalities , and 5 autonomous regions . four studies were conducted among all ages , with the age of participants in 9 studies being greater than 30 years old ; 3 studies did not provide information regarding the age range investigated . the sampling methods applied in the studies included random sampling , stratified sampling , cluster sampling , multistage - stratified sampling , or combinations of these and general surveys . in terms of diagnostic criteria for diabetes , 7 studies used the american diabetes association criteria ( ada ) from 1997 or 2009 , 31 studies used the criteria of the world health organization ( who ) from 1985 , 1988 , 1995 , or 1999 , one study used the international diabetes federation criteria ( idf ) from 2005 , and one study was performed according to the criteria outlined in the lanzhou conference on diabetes in china ; however , 12 studies did not provide clear information on the diagnostic criteria employed . eighteen studies used fasting plasma glucose ( fpg ) and oral glucose tolerance tests ( ogtt ) as the methods of diagnosis , 17 studies used fpg alone , 13 studies used ogtt , and one study used fpg , ogtt , and urine glucose tests . the prevalence of diabetes in 56 studies varied from 0.61% to 20.85% . levels of awareness , treatment , and control ranged from 28.50 to 62.54% , 17.72 to 92.50% , and 6.86 to 35.87% , respectively ( table 1 ) . as shown in table 2 , the overall prevalence of diabetes was 6.41% ( 95%ci : 5.507.33 ) . table 2 and figure 1(a ) illustrate the trend in the overall prevalence of diabetes in mainland china from 1979 to 2012 , showing that it increased as time progressed . the lowest prevalence of 0.81% was found in 1979 initially and showed a stable increase until 2001 . after a slight decrease , the prevalence increased quickly from 2002 to 2009 ( with the highest prevalence of 15.60% in 2009 ) , before decreasing rapidly again from 2009 to 2012 . the prevalence of diabetes in males was 6.91% ( 95%ci = 5.728.09 ) , and the prevalence in females was 6.43% ( 95%ci = 5.127.74 ) ; there was no significant difference in the prevalence of diabetes between males and females ( or = 1.07 , 95%ci = 0.981.16 ) . overall , trends in the prevalence of diabetes between males and females were increased and similar to the overall trend for the prevalence of diabetes ; no significant differences could be observed between males and females ( figure 1(b ) ) . the prevalence of diabetes in urban and rural areas was 7.82% ( 95%ci = 5.939.72 ) and 6.26% ( 95%ci = 4.258.27 ) , respectively . a statistically significant difference could be found in the prevalence of diabetes between urban and rural areas ( or = 1.61 , 95%ci = 1.252.06 ) . as shown in figure 1(c ) , trends in the prevalence of diabetes in both urban and rural areas increased over time , and the prevalence of diabetes in urban areas was consistently higher than that in rural areas . the prevalence of diabetes in the groups aged less than 39 , 4059 , and over 60 was 1.98% , 6.96% , and 13.24% , respectively . table 2 and figure 1(d ) show the information and trends in the prevalence of diabetes by age . an increasing tendency could be observed in all age groups over time . also , the prevalence of diabetes increased with age ( figure 1(d ) ) . however , no obvious increasing trend could be observed in awareness of diabetes from 1998 to 2012 ( figure 2(a ) ) . overall , the rate of awareness of diabetes in males was 40.86% and was 41.58% for females . no significant difference could be found in the awareness of diabetes between males and females ( or = 0.99 , 95%ci = 0.871.13 ) ( table 3 ) . the awareness rate in urban areas was 44.25% ( 95%ci = 32.655.9 ) , while it was 34.27% ( 95%ci = 2147.54 ) in rural locations ( table 3 ) . the combined result for the treatment rate of diabetes was 42.54% ( 95%ci = 13.6971.38 ) ( table 3 ) . as shown in figure 2(b ) , an increasing tendency in the treatment of diabetes could be observed from 2001 to 2005 . however , it decreased quickly after 2008 and then rapidly increased again from 2010 to 2011 . the treatment rate of diabetes in males was 38.48% , whereas it was 41.18% for females . comparing the treatment rate of diabetes for males to that of females , no significant difference could be observed ( or = 0.9 , 95%ci = 0.721.14 ) ( table 3 ) . the treatment rates in urban and rural areas were 50.18% ( 95%ci = 31.22100.39% ) and 45.42% ( 95%ci = 12.7% and 103.6% ) , respectively ( table 3 ) . comparing the treatment rate of diabetes between rural and urban areas , there was no significant difference ( or = 1.30 , 95%ci = 0.772.20 ) . the pooled control rate was 20.87% ( 95%ci = 10.7630.97 ) for diabetes from the combined information of eight studies ( table 3 ) . there was an increasing tendency in the control of diabetes from 2001 to 2008 , whereas the trend of control from 2008 to 2011 was similar to that of the treatment ( figure 2(b ) ) . overall , the control rate of diabetes in males was 19.26% and was 19.03% for females . there was no significant difference between males and females ( or = 1.06 , 95%ci = 0.981.15 ) ( table 3 ) . the control rate of diabetes in urban areas was 16.7% ( 95%ci = 2.3935.8% ) and 18.59% ( 95%ci = 1.238.39 ) in rural locations . no significant differences were found between urban and rural populations ( or = 18.59 , 95%ci = 1.2038.39 ) ( table 3 ) . due to the rapid development of the economy in china , the dietary habits and lifestyles of individuals have changed remarkably , which might have contributed to the increase in many chronic diseases such as diabetes [ 11 , 80 ] . overall , the results of our study showed that the prevalence , awareness , treatment , and control of diabetes in mainland china were 6.41% , 45.81% , 42.54% , and 20.87% , respectively . a higher prevalence of diabetes was found in urban areas than in rural locations . as for the awareness of diabetes , this was also higher among residents in urban than in rural areas . there were no significant differences when data were stratified by gender or location in the subgroup analysis with regard to the treatment and control of diabetes . to the best of our knowledge , our study is the first systematic review on the awareness , treatment , and control of the diabetes and covers the longest time period from 1979 to 2012 in mainland china on the level of survey above the provincial . the diabetes prevalence in our study ranged from 0.61% to 20.85% with an average of 6.41% ; however , many authors have shown different findings when studying other countries in asia . similar to our results , it was reported that the prevalence of diabetes in indonesia is 5.7% . in contrast , a survey conducted in vietnam indicated a prevalence of 1.4% to 2.5% , which is relatively lower than that of our study . on the contrary , epidemiological data from thailand and japan show that diabetes prevalence estimates were approximately 11.9% and 10% , which were higher than our study [ 82 , 83 ] . the wide variation in the prevalence could be explained by the heterogeneity of the studies , geographical differences , and the application of different diagnostic and sampling methods . according to the statistics of the idf , nationwide epidemiological surveys of diabetes were conducted in china and the prevalence showed a 5-fold increase [ 27 , 32 , 8587 ] . in accordance with this , an increasing trend in the prevalence of diabetes was found in our study . however , limited studies were included for pooled prevalence estimates ; therefore , more studies with high quality and larger sample sizes are required to further confirm our findings . as to the prevalence by gender , no significant difference could be found in our study between males and females , which is similar to the results of most studies according to the international diabetes federation ( idf ) . however , the study reported by yang et al . in 2010 showed a difference in the prevalence of diabetes between males and females . this might partly be explained by the fact that men are more accustomed to unhealthy diets due to their work and also smoke more frequently than women , which are the main risk factors for diabetes . moreover , men are much more careless with regard to their health . however , further studies are required to confirm our findings . in the analysis stratified by location , the prevalence of diabetes in urban areas was higher than in rural locations , with an odds ratio of 1.61 for residents in urban and rural areas , which suggests that residents in urban areas were more likely to suffer from diabetes than those in rural regions . in accordance with our findings , many studies worldwide have also reported a higher prevalence of diabetes in urban areas than in rural populations [ 32 , 86 , 88 ] . with the rapid development of the economy urbanization is related to the change in the food supply and lifestyles which could lead to an unhealthy diet , sedentary habits , and overnutrition [ 72 , 90 ] . the prevalence of overweight and obesity has increased over the past two decades , especially in urban areas and high - income groups , which are the risk factors for diet - related disease and contribute to the development of diabetes . it was reported that the prevalence of diabetes increased with age [ 85 , 86 ] . a nationwide epidemiology survey showed that prevalence of diabetes in the age group of 2039 was 1.01% , while it reached 6.31% in the age group over 40 ; however , the highest prevalence of 11.34% could be found in the age group over 60 . results of our study further confirm these findings . with the largest population in the world , china has become an ageing country ; therefore , the prevalence of diabetes will increase as a result of the aging population in china . ten studies provided information on the awareness of diabetes . among the included study subjects , 45.81% were aware of having diabetes . from 1998 to 2011 , there was no obvious improvement in the awareness rate of diabetes . however , we report a difference between the urban and rural areas when conducting stratified analysis by location . this difference in awareness may be a result of a lack of access to health care and knowledge in rural areas . increasing the awareness through health education remains an effective method for the prevention and treatment of diabetes . among the participants who had diabetes in our study , 42.54% of them were undergoing treatment this is high compared with the study which first reported the treatment rate of diabetes ( 27.2% ) in china . the results of our study also showed an increasing trend in the treatment rate of diabetes from 2001 to 2011 . convenient , economic , and effective detection tools could improve the treatment rate of this disease . it was reported that controlling blood glucose at a normal level can prevent many diabetes - related complications , such as retinopathy , nephropathy , neuropathy , and macroangiopathy . the control rate of diabetes in our study is 20.87% , which is higher than that reported from a previous study with a control rate of 9.7% , but lower than that identified in another study ( 40.3% ) . the control of diabetes can be influenced by many factors , for example , obesity , smoking , insufficient exercise , and genetic susceptibility . moreover , adjusting dietary habits , changing lifestyles , and giving up smoking are essential when taking the risk factors for diabetes into account . coory stated that it was very difficult to avoid heterogeneity in a meta - analysis , and this is also true for the meta - analysis of data from epidemiologic studies due to methodological problems . there were several methodological problems that might help to explain the heterogeneity : ( 1 ) different degrees of urbanization and socioeconomic conditions existed ; ( 2 ) the included studies were carried out in different cities at various time points ; ( 3 ) large differences existed in the age range , sampling methods , sample sizes , and response rates ; ( 4 ) different diagnostic criteria and diagnosis methods were used in studies , with other studies not mentioning them at all ; and ( 5 ) not all of the included studies provided sufficient information on gender , location , and age for subgroup analysis . there are some limitations of the current study which need to be highlighted . firstly , the present study might have underestimated the true prevalence of diabetes , since some of the studies included in the present review only used fasting plasma glucose ( fpg ) levels to diagnose new cases of diabetes ; also , some studies did not provide any information about the diagnostic methods . thus , more accurate measurements in the detection of undiagnosed diabetes are necessary in future research , for example , the two - hour glucose tolerance test and the oral glucose tolerance test ( ogtt ) . secondly , in addition to the subgroups of gender , location , and age , we did not report any positive risk factors associated with the prevalence of diabetes and the awareness , treatment , and control of the diabetes ( e.g. , overweight , obesity , hypertension , and hyperlipidemia ) ; thus , we were unable to assess the association between them . the age distribution in the sample population could markedly affect the results , since some locations contain a large number of individuals > 40 years old ; therefore , the prevalence of diabetes in some studies is likely to be higher than in others . fourthly , limited studies were included in our meta - analysis for the pooled estimates ; for example , only one study was included for analysis in 2009 and 2012 as studies from that time are possibly yet to be published . this could also be seen with regard to the trends in awareness of diabetes in 2001 and in the treatment and control of diabetes in 2011 . a further limitation of the present study is that information in the subgroup analysis of awareness , control , and treatment by gender , location , and age was limited ; therefore , we did not perform the relevant trend analyses . the study showed an increasing trend for the prevalence , treatment , and control of diabetes among the population of mainland china from 1979 to 2012 , but not in the awareness . this review will help us understand the gaps in the current research , which is useful for investigators and health care providers regarding aboriginal health . thus , urgent measures are needed to prevent the high prevalence of diabetes and to improve diabetes awareness , control , and treatment among the chinese populations .

objective . we reviewed a cohort of patients with previously untreated locoregional advanced head and neck squamous cell carcinoma ( hnscc ) who received a uniform chemoradiotherapy regimen . methods . retrospective review was performed of 105 patients with stage iii or iv hnscc treated at greater baltimore medical center from 2000 to 2007 . radiation included 125 cgy twice daily for a total 70 gy to the primary site . chemotherapy consisted of cisplatin ( 12 mg / m2/h ) daily for five days and 5-fluorouracil ( 600 mg / m2/20 h ) daily for five days , given with weeks one and six of radiation . all but seven patients with n2 or greater disease received planned neck dissection after chemoradiotherapy . primary outcomes were overall survival ( os ) , locoregional control ( lrc ) , and disease - free survival ( dfs ) . results . median followup of surviving patients was 57.6 months . five - year os was 60% , lrc was 68% , and dfs was 56% . predictors of increased mortality included age 55 , female gender , hypopharyngeal primary , and t3/t4 stage . twelve patients developed locoregional recurrences , and 16 patients developed distant metastases . eighteen second primary malignancies were diagnosed in 17 patients . conclusions . the crt regimen resulted in favorable outcomes . however , locoregional and distant recurrences cause significant mortality and highlight the need for more effective therapies to prevent and manage these events . diabetes mellitus ( dm ) is one of the primary causes of premature death and disability worldwide . we performed a systematic review and meta - analysis of the published literature regarding the trends in prevalence , awareness , treatment , and control of diabetes mellitus in mainland china . pubmed , embase , chinese biomedical database , china national infrastructure database , chinese wan fang database , and chongqing vip database were searched . fifty - six eligible studies were included . increasing trends in the prevalence , treatment , and control of diabetes in mainland china from 1979 to 2012 were observed . the pooled prevalence , awareness , treatment , and control of diabetes mellitus were 6.41% , 45.81% , 42.54% , and 20.87% , respectively . a higher prevalence of diabetes mellitus was found in urban ( 7.48% , 95%ci = 5.45~9.50 ) than rural ( 6.53% , 95%ci = 4.30~8.76 ) areas . furthermore , an increasing chronological tendency was shown in different subgroups of age with regard to the prevalence of diabetes . a higher awareness of dm was found in urban ( 44.25% , 95%ci = 32.60~55.90 ) than rural ( 34.27% , 95%ci = 21.00~47.54 ) populations , and no significant differences were found in the treatment , and control of diabetes among the subgroups stratified by gender and location . from 1979 to 2012 , the prevalence , treatment , and control of diabetes mellitus increased ; nevertheless , there was no obvious improvement in the awareness of diabetes .

focal segmental glomerulosclerosis ( fsgs ) is a clinicopathologic entity responsible for up to 20% of all cases of end - stage kidney disease in the united states . the incidence of fsgs is estimated at 7 per million individuals annually , has a slight male predominance ( male : female ratio 1.52 ) , and now represents the leading cause of idiopathic nephrotic syndrome in adults . although the pathogenesis of fsgs has not yet been fully described , the lesions that are induced include tuft collapse , segmental hyalinosis , and effacement of foot processes as revealed by electron microscopy . fsgs may also occur secondarily to certain genetic predispositions as well as disorders such as sickle cell disease , hiv nephropathy , and obesity ; in these instances , plasma exchange would not be considered a primary therapeutic option and treatment would instead be directed at the underlying driver . clinically , patients with fsgs manifest significant , nephrotic - range proteinuria , reduced renal function , and hypertension . eighty percent of fsgs cases are characterized as primary and are suspected to be caused by a circulating fsgs permeability factor , a low - molecular weight anionic protein . this same factor may be responsible for the recurrence of fsgs in the renal allograft , a scenario that occurs in approximately 2050% of primary allografts , most commonly within the first 3 months following transplant [ 4 , 5 ] . plasmapheresis removal of this factor appears to effectively treat many such cases [ 6 , 7 ] . recurrent fsgs can be suspected in patients who develop impaired graft function and significant proteinuria , defined as > 1 g/24 h in the posttransplant period . in many cases early recognition is therefore critical in properly diagnosing and managing this disorder . if recurrent fsgs is suspected ( even in the absence of a pretransplant history of fsgs ) then it is important to inform the pathology department of this suspicion so that biopsy specimens can be properly processed for electron microscopy , which is needed to evaluate for the foot process effacement characteristic of fsgs . the american society for apheresis guidelines designate recurrent fsgs in the renal allograft as a category i indication for therapeutic plasma exchange ( grade 1b , strong recommendation ) . we present two patients who developed recurrences of fsgs in their primary renal allografts treated with plasmapheresis and intravenous immune globulin ( ivig ) . a 39-year - old hispanic male with a history of end - stage renal disease ( esrd ) , hypertension and hypertrophic cardiomyopathy underwent deceased donor renal transplantation with thymoglobulin induction and standard steroid taper . he developed poor graft function and nephrotic - range proteinuria beginning on posttransplant day 1 . he required posttransplant hemodialysis on posttransplant days 3 , 6 , 8 , and 11 , as well as initiation of therapeutic plasma exchange and ivig on postoperative day 5 . renal transplant biopsies obtained on posttransplant day 7 confirmed the team 's suspicion for fsgs recurrence in the renal allograft . the patient is currently 4 years posttransplant and continues to demonstrate excellent renal allograft function ( serum creatinine of 1.6 ) with continued absence of proteinuria . a 37-year - old asian male with esrd due to primary fsgs underwent living unrelated renal transplantation performed with thymoglobulin induction followed by standard steroid taper . on posttransplant day 3 , significant proteinuria and a rise in serum creatinine developed . therapeutic plasma exchange was initiated on posttransplant day 4 and continued forward as noted in figure 3 . the patient also received a single dose of rituximab as well as ivig as treatment for his recurrent fsgs . a kidney biopsy on posttransplant day 8 was negative for rejection , but demonstrated typical foot process effacement changes consistent with fsgs recurrent in the renal allograft . following an initial complete response to therapeutic plasma exchange by posttransplant day 78 , a repeat allograft biopsy was obtained , which demonstrated improvement in the degree of foot process effacement and no evidence for rejection . the second bout of allograft dysfunction was ultimately discovered to be due to interval development of high - grade stenosis in the allograft ureter leading to a postobstructive nephropathy . as of posttransplant day 317 , blood urea nitrogen and creatinine ( both as mg / dl ) are 13 and 1.3 , respectively , and there is continued absence of proteinuria . we describe two cases of fsgs recurrent in the primary allografts successfully treated with therapeutic plasma exchange . in both cases , remission was associated with resolution of proteinuria and histologic improvement in the degree of foot process effacement on electron microscopy . , 10/11 patients resulted in a persistent reduction in 24-hour urine protein following 511 ( mean of 6 ) therapeutic plasma exchange procedures in conjunction with immunosuppressant medications . in another study , patients with recurrent fsgs not managed with plasma exchange were used as historic controls and were compared to 13 patients treated with plasma exchange . while persistent proteinuria contributed to graft loss in all untreated controls , 11/13 ( 85% ) patients treated with plasma exchange achieved either complete or partial remission . in another report , three of five patients with early recognition of recurrent fsgs treated with plasma exchange responded well , resulting in the reversal of foot process effacement and remission of proteinuria . among 14 pediatric patients with fsgs recurrent in the renal allograft , 9 were treated with 10 cycles of plasma exchange ( 3 cycles per week , the same regimen as ours ) with 5/9 ( 55.5% ) and 1/9 ( 12% ) achieving complete and partial remissions , respectively ; no remissions were observed among the 5 patients not treated with plasma exchange . in a treatment series of fsgs recurrent in the renal allograft , alachkar and colleagues noted a complete response rate in 9/25 ( 36% ) and partial response in 10/25 ( 25% ) of patients treated with combinations of plasmapheresis and rituximab . interestingly , levels of soluble urokinase - type plasminogen activator ( supar ) a prospective permeability factor elevated in up to two thirds of those with primary fsgs were found to correlate both with recurrence and response [ 11 , 12 ] . supar , however , is not specific for fsgs and has shown to be elevated in immune activation states , such as sepsis , hiv infection , and paroxysmal nocturnal hemoglobinuria , among others . furthermore , supar rebounds quickly following plasma exchange , and clinical response associated with plasma exchange treatment occurred despite sustained reductions in supar levels . . showed no improvement in proteinuria upon addition of rituximab to chronic plasma exchange therapy in three retransplanted patients with recurrent fsgs . in contrast , kumar and colleagues , upon retrospective review , identified complete responses [ defined as reduction in urine protein : creatinine ( pr : cr ) ratio to < 0.2 ] and partial responses ( defined as decrease in urine pr : cr ratio by 50% and < 2.0 ) among 2/8 and 4/8 pediatric patients with recurrent fsgs . there have also been cases of responses to rituximab after incomplete or absent response to plasma exchange [ 18 , 19 , 20 ] . the precise mechanism of rituximab , a monoclonal antibody directed against cd20 , merits further research . a cd20-independent mechanism was studied by fornoni and colleagues , who described marked disruption of the actin cytoskeleton of podocytes exposed to fsgs sera . rituximab , through binding and preservation of sphingomyelin - related enzymes ( sphingomyelin phosphodiesterase acid like 3b - smpdl-3b ; acid sphingomyelinase ) , partially prevented the cytoskeletal damage incited by fsgs sera , thus protecting the podocyte filtration barrier and preserving function . this mechanism was corroborated by takahashi and colleagues , who demonstrated that rituximab , but not purified igg , significantly reduced proteinuria in an adriamycin - induced nephropathy rat model . this appears to be related to binding of rituximab to glomerular smpdl-3b and protection of podocyte - related proteins , nephrin and podocin . although precise mechanisms require further research to elucidate , the authors provide a theory that smpdl-3b is a key factor in the actin - cytoskeleton remodeling process within podocytes and that rituximab binding to smpdl-3b provides a salutary effect . evidence appears to support a similar protective action of cyclosporine a on podocytes , which blocks calcineurin - mediated desphosphorylation of synaptopodin , thus preserving cytoskeleton stability and preventing foot process effacement . there is also evidence of reduction in proteinuria with higher - dose intravenous ( but not standard - dose oral ) cyclosporine . inhibition of the renin - angiotensin system ( ras ) also has an antiproteinuric effect in recurrent fsgs patients . in one report , authors used triple ras suppression with angiotensin - converting enzyme inhibitor , angiotensin receptor blocker , and aliskiren , a direct renin inhibitor . their patient was treated with a combination of rituximab and plasma exchange , and the sequential triple ras inhibition by the latest addition of aliskiren led to a significant reduction in recalcitrant proteinuria . in a study by gohh et al . , preemptive plasma exchange treatment was explored as a means of reducing posttransplant fsgs recurrence in ten patients considered at high risk for recurrence ( esrd within 3 years of fsgs diagnosis or advanced azotemia at presentation or prior allograft loss due to recurrent fsgs ) . at a mean of 751 314 days three patients , each of whom had already manifested previous graft loss due to recurrent fsgs , developed biopsy - proven recurrence in the allograft . two of these patients returned to dialysis ; the third remained nephrotic with a creatinine of 2.6 mg / dl at the time of publication . park and colleagues , in an observational cohort study , found no difference in fsgs recurrence rates whether adult kidney transplant patients received pretransplant plasma exchange ( 1/4 , 25% ) , pretransplant plasma exchange plus rituximab ( 1/5 , 20% ) or no pretreatment ( 5/18 , 27.7% ) . in the pediatric population , gonzalez and colleagues found absent protective effect of pretransplant plasma exchange with recurrence rates of 47 and 63% , respectively , for living and deceased donor first - time kidney transplantation . however , preemptive treatment did lead to a lower than expected rate of recurrence among patients undergoing transplant who had previously lost a graft due to recurrence . among those with recurrences , plasma exchange was highly effective as a treatment modality with 88% complete or partial remission rate . in patients referred for renal transplantation , a vague diagnosis of hypertensive nephrosclerosis particularly in younger patients raises concern for the absence of a clearly defined etiology for primary renal failure . in some cases , a primary disorder such as fsgs may have been causative . this is of particular concern considering the high risk of recurrence of primary fsgs in the renal allograft . fsgs recurrent in the renal allograft can be diagnosed on electronic microscopy in proteinuric patients . the prompt institution of therapeutic plasma exchange is an important treatment modality , and rituximab may be added to this as adjunctive therapy . further research is required regarding the role of rituximab therapy either as a therapeutic modality in established fsgs recurrence or possibly as pretransplant prophylaxis to reduce the risk of recurrence . monitoring of treatment response to ongoing plasmapheresis treatment is accomplished through measurement of urine spot pr : cr ratios and serum creatinine . in responding patients , treatment should be continued until proteinuria has been reliably suppressed to below 0.5 g / day . was in part supported by grants from nih - ncrr ul1 tr000153 , kl2 tr000147 , and the juvenile diabetes research foundation international 17 - 2011 - 609 . key goals of health systems are to improve the health of the population and to do so in an equitable way . although the health of the population to a large extent is influenced by broader improvements in living conditions , well - functioning health care systems also contribute to the health of populations . however , their ability to do so varies and there is increasing interest in measuring their performance ; the introduction and diffusion of medical innovations should be an important part of this process . there have long been concerns about the delay in introducing innovative drugs in different countries , . for instance , this has been reported to have been somewhat delayed in the united states , sweden and italy when compared with united kingdom , west germany and france , . however , given the relative ease in obtaining data on year of registration , delays in the registration process have been the main focus of debate about international variation in the timing of introduction of innovative drugs , even though other health system factors have also been identified , . in the european union ( eu ) however , for a drug to impact on the health of the population , it must also be used in clinical practice . for instance , pharmaceuticals may have been used in clinical trials before registration year ( which may have a considerable impact if trials are relatively large and the condition is rare ) , or there may have been a delay after the registration year before the drug is widely adopted . the theory of diffusion of innovations envisages cumulative adoption following an s - shaped curve , with rogers describing diffusion of innovations as the process through which an innovation is communicated through certain channels over time among the members of a social system . ( 1 ) innovators , about 2.5% of the population. they are not opinion leaders , but prone to novelty and with little to lose . ( 2 ) thereafter come : ( 3 ) early majority , who tend to rely heavily on the early adopters and ( 4 ) the late majority , which relies in turn on the early majority . they are considered more conservative and will not adopt an innovation until it is standard practice . finally , ( 5 ) the laggards , ( or traditionalists ) , wedded to the old ways , are critical of new ideas and will only accept them once the new idea has become mainstream or even tradition . commonly , these are descriptive studies showing the variation in the time of introduction of new innovations . other studies analyse factors that could explain the process of diffusion . furthermore , there has been concerns about inequity in medical care related to variations in the diffusion of innovation to different population groups such as racial and ethnic disparities 1215 as well as gender , age and social differences , . these studies have used a variety of empirical data such as health administrative data , , medical records or questionnaires to or interviews with key informants 2325 . for pharmaceuticals , official data on registration as well as sales and prescribing statistics of pharmaceuticals , , applying the theory of diffusion to the introduction of new pharmaceuticals implies the need to obtain empirical data showing when the innovators and early adaptors started to use the new treatment . however , where our goal is to identify when an innovation might be expected to improve the health of the population , we will need data also on the diffusion of the innovation . this study is part of the amiehs project ( avoidable mortality in the european union : towards better indicators for the effectiveness of health systems ) , which seeks to develop indicators of the effectiveness of health systems . mortality for specific causes of death that are considered amenable to medical care is increasingly used as an indicator of the quality of health care . high death rates from these causes , collectively termed avoidable or amenable mortality , have been suggested to motivate further investigations of the quality of health care . there is now extensive experience in their use in different countries , health administrative areas , socioeconomic and ethnic groups and with respect to gender , . several studies have shown that mortality from these causes have decreased more rapidly than total mortality in several industrialized countries 3032 . for some avoidable causes of death , mortality has also been found to decline faster after the introduction of innovations in health care , such as improved management of hypertension , leading to lower mortality from cerebrovascular disease and screening to prevent deaths from cervical cancer . however , to assess the effectiveness of health care at a population level , a precise measure of the timing of the introduction of innovations is necessary in order to evaluate whether they have impacted on the health of the population . the objective of this paper is to analyse the variation in the introduction of specific pharmaceutical innovations that may influence the mortality outcome among seven european countries for comparison applying both official registration data and indicators of the start of the diffusion of the innovative drugs . key goals of health systems are to improve the health of the population and to do so in an equitable way . although the health of the population to a large extent is influenced by broader improvements in living conditions , well - functioning health care systems also contribute to the health of populations . however , their ability to do so varies and there is increasing interest in measuring their performance ; the introduction and diffusion of medical innovations should be an important part of this process . there have long been concerns about the delay in introducing innovative drugs in different countries , . for instance , this has been reported to have been somewhat delayed in the united states , sweden and italy when compared with united kingdom , west germany and france , . however , given the relative ease in obtaining data on year of registration , delays in the registration process have been the main focus of debate about international variation in the timing of introduction of innovative drugs , even though other health system factors have also been identified , . in the european union ( eu ) however , for a drug to impact on the health of the population , it must also be used in clinical practice . for instance , pharmaceuticals may have been used in clinical trials before registration year ( which may have a considerable impact if trials are relatively large and the condition is rare ) , or there may have been a delay after the registration year before the drug is widely adopted . the theory of diffusion of innovations envisages cumulative adoption following an s - shaped curve , with rogers describing diffusion of innovations as the process through which an innovation is communicated through certain channels over time among the members of a social system . ( 1 ) innovators , about 2.5% of the population. they are not opinion leaders , but prone to novelty and with little to lose . ( 2 ) thereafter come : ( 3 ) early majority , who tend to rely heavily on the early adopters and ( 4 ) the late majority , which relies in turn on the early majority . they are considered more conservative and will not adopt an innovation until it is standard practice . finally , ( 5 ) the laggards , ( or traditionalists ) , wedded to the old ways , are critical of new ideas and will only accept them once the new idea has become mainstream or even tradition . commonly , these are descriptive studies showing the variation in the time of introduction of new innovations . other studies analyse factors that could explain the process of diffusion . furthermore , there has been concerns about inequity in medical care related to variations in the diffusion of innovation to different population groups such as racial and ethnic disparities 1215 as well as gender , age and social differences , . these studies have used a variety of empirical data such as health administrative data , , medical records or questionnaires to or interviews with key informants 2325 . for pharmaceuticals , official data on registration as well as sales and prescribing statistics of pharmaceuticals , , applying the theory of diffusion to the introduction of new pharmaceuticals implies the need to obtain empirical data showing when the innovators and early adaptors started to use the new treatment . however , where our goal is to identify when an innovation might be expected to improve the health of the population , we will need data also on the diffusion of the innovation . this study is part of the amiehs project ( avoidable mortality in the european union : towards better indicators for the effectiveness of health systems ) , which seeks to develop indicators of the effectiveness of health systems . mortality for specific causes of death that are considered amenable to medical care is increasingly used as an indicator of the quality of health care . high death rates from these causes , collectively termed avoidable or amenable mortality , have been suggested to motivate further investigations of the quality of health care . there is now extensive experience in their use in different countries , health administrative areas , socioeconomic and ethnic groups and with respect to gender , . several studies have shown that mortality from these causes have decreased more rapidly than total mortality in several industrialized countries 3032 . for some avoidable causes of death , mortality has also been found to decline faster after the introduction of innovations in health care , such as improved management of hypertension , leading to lower mortality from cerebrovascular disease and screening to prevent deaths from cervical cancer . however , to assess the effectiveness of health care at a population level , a precise measure of the timing of the introduction of innovations is necessary in order to evaluate whether they have impacted on the health of the population . the objective of this paper is to analyse the variation in the introduction of specific pharmaceutical innovations that may influence the mortality outcome among seven european countries for comparison applying both official registration data and indicators of the start of the diffusion of the innovative drugs . these were selected on the basis of their effectiveness , as demonstrated in clinical trials , in reducing specific causes of death , where those causes were sufficiently common and where there had been an observed decline in mortality in recent decades , making it possible to undertake robust statistical testing for any association in another part of the project . interventions included in the analysis of timing of innovations cisplatin was introduced for the treatment of testicular cancer already in the 1970s and has been shown to improve outcome when included in combination therapy . the h2-receptor antagonist cimetidine was evaluated in clinical trials of peptic ulcer treatment in the late 1970s supporting evidence on the ulcer healing , . tamoxifen has been shown to reduce recurrences and mortality from breast cancer in several clinical trials . clinical trials have found oxalaplatin to be effective as a component of combination therapy for advanced colorectal cancer , . the antiretroviral drugs azidothymidine and zidovudine were shown to improve survival with aids in randomized clinical trials for these specific innovations , a questionnaire was developed and answered by the participating partners from the united kingdom , the netherlands , germany ( west germany ) , france , spain , estonia and sweden . information was sought on the first introduction of the innovation and the period of implementation . for the first part , documents on the official and organized introduction were asked for . these were for instance , the registration year for pharmaceuticals as well as the year of national programs and guidelines . to get information on the implementation of the interventions , data on scientific or committee reports evaluating the implementation were requested . for pharmaceuticals , this data was used to verify that the medicine has been available in the respective country and also to indicate the speed of the implementation . to facilitate comparisons between countries , statistics on daily defined doses ( ddd ) sales statistics were available from sweden ( 19772008 ; only from year of registration ) , estonia ( 19992008 ) , germany ( 19992008 ; hospital prescribing and privately insured patients were unavailable ) ) france ( 20022007 ) , united kingdom ( mainly 1999 ; hospital prescribing unavailable ) and spain ( 20002008 ; hospital prescribing unavailable ) . sales statistics measured by ddd was available for most of the period from sweden , estonia and germany only . articles registered in medline with any of the key words ( or words in title or abstract ) standards , guidelines , official policy , consensus development , evaluation studies or clinical trial in combination with the terms for the innovation and the country names were scrutinized for applicable information . the country representatives were asked to comment on the data found and to collect further data based on the information . the data from the questionnaire and literature review were combined in order to get several indicators of the diffusion process of the specific innovations in each country . we looked for both very early indicators of early adaptors introducing the method and indicators of a continued diffusion of the method and used specific criteria for defining the start of the diffusion criteria 1 : the first documented year of introduction of the pharmaceutical in a specific country was used to define the year of the start of diffusion . the start of first clinical trials or clinical studies , available sales statistics from the introduction period or evaluation reports describing the introduction of the pharmaceuticals were accepted as data on the start of the diffusion . if the starting year of the clinical trial was not mentioned , we used the year 3 years before the publication year as the indicator of the introduction . when no data was available , the year of registration was used . criteria 2 : in order to define the start of the diffusion , we also judged that data indicating a continued diffusion of the drug was needed . this was indicated by further clinical trials , evaluation reports , guidelines and sales statistics . we present in this article the references to the early indicators of the start of diffusion and to evaluation reports and guidelines indicating the further diffusion of the pharmaceuticals . the availability of sales statistics was presented above . when data was available , we analysed the year when the peak level of sales of the pharmaceutical occurred and the diffusion time from start of diffusion to this year . the variation in year of introduction among the countries was studied using both official data of year of registration and data on the start of the diffusion of the pharmaceuticals , respectively . the time lag between the first introduction in any of the countries and in the others was calculated . a mean score of the time lag including data on all the six pharmaceuticals was analysed for each country . with the exception of estonia , cisplatin was introduced for the treatment of testicular cancer in all countries in the 1970s or early 1980s ( table 2 ) . in estonia , it was delayed until the 1990s , when the country had achieved independence . in most countries , the introduction started with clinical trials or other clinical studies in the period 19761981 4347 , while registration was some years later , that is , between 1979 and 1983 . in several countries , cisplatin was registered for the treatment of cancers at several other sites so sales data could not be used to evaluate its diffusion as a treatment for testicular cancer . one study from the netherlands found that cisplatin was not in widespread use between 1970 and 1978 ( table 3 ) . in the united kingdom , the uk children 's cancer study group , followed up children with testicular cancer from 1979 to 1988 , during which period only about 7 % of patients received the drug . the year of the introduction of medical innovations measured by indications of the start of diffusion of innovations compared to the registration year for selected causes of death doi , indication of the start of the diffusion of innovation ; n.d . , no data available ; reg . , based on official data of registration . guidelines and evaluation reports indicating the continued diffusion of medical innovations cimetidine was introduced for the treatment of peptic ulcer in most countries in the late 1970s or early 1980s . in estonia however , the drug was first registered in 1993 . in germany and france , , clinical trials started a few years before the year of registration . in spain , the year of registration and first clinical trial coincided tamoxifen , used to treat breast cancer , was introduced in most countries in the late 1970s or early 1980s . however , in estonia , it was first available in the 1990s , where it was registered in 1992 . in germany , clinical trials were reported a few years before registration , and in sweden , the first reported clinical trials , coincided with registration . immunosuppressive treatment with cyclosporine following kidney transplantation was introduced in the 1980s in most countries with the exception of estonia where it was again delayed until the 1990s . in most countries , the year of registration and the start of the clinical trials were close in time . however , in germany , clinical trials were reported 11 years before the official registration . clinical trials were reported from united kingdom , , the netherlands , germany , spain and sweden . as with cisplatin , clinical trials of oxalaplatin started before registration ( which was usually at the end of the 1990s ) in several countries . the time lag varied between 4 years in the united kingdom and spain to 8 years in france . in some countries , the diffusion process can be assessed by consensus statements and guidelines . in the united kingdom , guidelines were published in 2002 , 7 years after the introduction oxalaplatin was recommended for patients with inoperable liver metastases to make them eligible for surgery . in germany , a 2000 consensus statement on treatment of colon cancer mentioned oxalaplatin as a promising new drug but not yet generally recommended . in sweden , oxalaplatin was recommended as one alternative in an evidence review from 2001 , that is , 13 years after the introduction . the antiretroviral drugs azidothymine or zidovudine were introduced in most countries between 1987 and 1995 . in the netherlands , the registration year coincided with the first reported clinical trial starting in 1987 . in the united kingdom , , germany , france , spain and sweden , clinical trials started a few years after the year of registration . their use was endorsed by clinical guidelines or recommendations in united kingdom , germany , spain and sweden . the guidelines were published between 7 and 17 years after the introduction in the respective country . as illustrated in table 4 , the variation among countries in the introduction of new drugs was largest for cisplatin , cimetidine and tamoxifen , all introduced first in the 1970s . for the most recent pharmaceuticals , the antiretroviral drugs and oxalaplatin , the variation was considerably less . some countries were consistently among the first to introduce new drugs , such as germany , france , sweden , united kingdom and the netherlands . introduction of modern drugs was delayed until after independence in estonia , while spain often introduced the drugs studied later than other countries . variation in the start of diffusion of medical innovations among countries based on country - specific year of introduction the delay since the first introduction was shortest for france , the netherlands and united kingdom ( table 5 ) , and longest for estonia . this was true both when the official year of registration and when other data on the start of the diffusion of innovation were used . when the start of diffusion was used the variation was greater and the ranking between the countries differed somewhat from what was found using the registration year . for instance , the delay in registration was shortest in the netherlands ( 1.3 years ) while the time lag until the start of diffusion was shortest in france ( 2.2 years ) . the sum * and mean scores of time lag in year of introduction after the corresponding year of introduction in the first of the countries for the six studied pharmaceuticals sum score , total lag compared with first country , six pharmaceuticals . n.a . , not applicable . for several innovations , the length of period of diffusion from introduction to the year when the peak level of use was reached was similar ( table 6 ) . for some pharmaceuticals cisplatin , cimetidine and cyclosporine a delay in peak level was found in estonia although when the time length for diffusion was similar . in these cases , the diffusion started later in estonia , but this seems however to have been compensated by a faster diffusion ( 11 years compared with about 20 years in the compared countries ) . for treatment with cisplatin , the early introduction in sweden was followed by a fast diffusion , which increased the variation among the countries further . for zidovudine , the diffusion seems to have been fast since the sales of the pharmaceutical peaked before or at the start of the period of available sales statistics in most countries , that is , around the late 1990s . the diffusion period * of selected medical innovations period between the year of the first indication of the start of diffusion ( as presented in table 2 ) and the year of the peak of sales level of the pharmaceutical in each country . several pharmaceuticals developed in the 1970s and 1980s were introduced about the same time in most countries with the exception for estonia , where cimetidine , cisplatin and cyclosporine were only introduced in the 1990s . this was true also for treatment of breast cancer with tamoxifen , although the variation was fairly large also among the other countries . for the drugs developed later , oxalaplatin and antiretroviral drugs , however , introduction was at about the same time in all countries , in the late 1980s or early 1990s . these results are consistent with the aspiration by the european countries to harmonize regulations with regards to medicines and other pharmaceutical products . the concept of the establishment of the multi state licensing procedure in 1975 was that pharmaceuticals that had been approved in one member country should be authorized in other countries . this procedure , which was not very successful at first , was replaced in 1987 by the concertation procedure , which in turn was replaced in 1995 by the european medicines evaluation agency . once estonia regained its independence , it became integrated with the global economy , especially since acceding to the eu in 2004 , and has seen sustained improvements in health care , . in general , innovative drugs were first introduced in the netherlands , the united kingdom and in france , whether assessed by date of registration or other indicators of when diffusion started . the process was generally delayed in spain , especially when indicators of when diffusion started were considered . the delay in sweden was somewhat larger when assessed by the start of diffusion than registration . importantly , these findings demonstrate that registration does not always coincide with uptake of drugs , even though most attention so far has been on the former , . thus , it is necessary to look at other characteristics of the health system to explain why some european citizens wait longer to receive innovative treatments than others , , especially given the success of harmonization of the registration process . of the more recently introduced pharmaceuticals ; oxalaplatin was first approved in france in 1996 , and approved throughout the eu through the mutual recognition procedure in 1999 , france being the reference member state . zidovudine was first approved in 1987 using national licenses , before the concertation procedure became mandatory for hiv / aids treatments . for these pharmaceuticals , the variation in timing in introduction was low among the countries , including estonia . for tamoxifen , there was a time lag in several countries between the registration of the drug and its use in clinical trials . at that time , several therapeutical strategies were being discussed and its role of tamoxifen seems not to have been clearly defined . scientific papers at that time argue that it had a limited role and called for more clinical trials , which started in most of the countries in this study in the late 1970s and 1980s . to define the start of the diffusion process , one needs empirical longitudinal data on the uptake of drugs , such as sales statistics . however , in most cases , these were only available for recent years when the drugs were already registered for some years . thus , sales statistics were mainly used to verify that the drug had diffused . instead , diffusion was often considered to have begun with the first clinical trials in each country , provided indicators of a continued diffusion were available . for tamoxifen and antiretroviral drugs , there may also have been less well documented use of the drug in some countries not captured by the search for clinical trials so the variation between countries should therefore be interpreted with caution . there were several indications of a continued diffusion of the different innovations . for several innovations , the length of period of diffusion seems to be similar among the countries according to sales statistics . for these pharmaceuticals , variations in use may instead reflect variation in the start of the diffusion among the countries . in most cases , there was a time lag of between 7 and 17 years from the introduction until guidelines were published . thus , the presence of clinical guidelines would not reflect an early phase of the diffusion of innovation promoted by innovators and early adaptors . a previous review have found that the mean compliance rate for guidelines was 54% 3 years after the publication . thus , it seems as if the publication of guidelines may reflect the phase when the innovation diffuses from the early majority to the late majority . this would be in line with the theory of diffusion of innovation , where the late majority is considered to be more conservative and will not adopt an innovation until it is standard practice . several modifiable reasons have been found for not using proven pharmaceutical interventions , including clinician , patient and system factors . thus , it may be possible to influence the implementation of a pharmaceutical innovation in order to limit the variation in introduction and diffusion . in order to establish a new medical drug in medical practice , its efficacy must be proved in clinical trials and the drug must be approved by the regulatory authorities based on data on efficacy and safety . efficacy refers to the potential of improving health of the patients under ideal conditions of use , which should be the case in clinical trials . the pharmaceuticals studied in this paper have all passed this phase of assessment and have been approved for use in medical practice . however , we must also consider that the effectiveness , defined as what is achieved under ordinary conditions , may vary . there may be variation in the uptake of pharmaceuticals and other medical interventions and the quality of care may differ . thus , the outcome of medical care may differ from what would be expected from the results of clinical trials . as pointed out by brook and lohr , there is need to integrate the concepts of efficacy , effectiveness , variations in population - based rates of use and quality of care when building models for policy , planning and evaluation of medical care . in order to assess the impact on populations health of a new medical drug , we have to be able to measure the timing of introduction of the new drug as well as the diffusion process . this information should then be linked to population outcome measures , in order to assess whether the new treatment has been effective in improving the health of the population . although the introduction of a new drug may be regarded as the date of registration , this paper has shown that the year when the drug was first used in medical care often differs from the official registration year . according to the theory of diffusion of innovations , the medical intervention may be introduced by innovators but in order to have a broader impact on populations health , there must also be a continued diffusion of the innovation . when applying this concept , we found that the registration year of the pharmaceuticals studied may differ from the year of introduction with a range between 11 years before and after the registration year . in assessing any impact on population health however , although we used a wide range of data in order to ascertain the introduction and diffusion of new innovations , our main finding is the need for harmonized data in eu countries . the lack of data on different phases of the diffusion process limited the possibilities to analyse the impact of introduction and diffusion of the new drugs . data on the diffusion of drugs and not simply the registration year should routinely be documented in a comparable way across the eu . this is most likely going to require prescribing data , which at present is inconsistent and only some elements are in the public domain . this should , ideally , include information on the indications for prescribing , although clearly , this will not be possible for some time , except where functioning patient information systems at a population level exist . it will also be important to use harmonized measures of the amount of drugs prescribed , such as the ddd . such improvements in the information on the introduction and diffusion of drugs would be a valuable tool in evaluating and planning health care . several pharmaceuticals developed in the 1970s and 1980s were introduced about the same time in most countries with the exception for estonia , where cimetidine , cisplatin and cyclosporine were only introduced in the 1990s . this was true also for treatment of breast cancer with tamoxifen , although the variation was fairly large also among the other countries . for the drugs developed later , oxalaplatin and antiretroviral drugs , however , introduction was at about the same time in all countries , in the late 1980s or early 1990s . these results are consistent with the aspiration by the european countries to harmonize regulations with regards to medicines and other pharmaceutical products . the concept of the establishment of the multi state licensing procedure in 1975 was that pharmaceuticals that had been approved in one member country should be authorized in other countries . this procedure , which was not very successful at first , was replaced in 1987 by the concertation procedure , which in turn was replaced in 1995 by the european medicines evaluation agency . once estonia regained its independence , it became integrated with the global economy , especially since acceding to the eu in 2004 , and has seen sustained improvements in health care , . in general , innovative drugs were first introduced in the netherlands , the united kingdom and in france , whether assessed by date of registration or other indicators of when diffusion started . the process was generally delayed in spain , especially when indicators of when diffusion started were considered . the delay in sweden was somewhat larger when assessed by the start of diffusion than registration . importantly , these findings demonstrate that registration does not always coincide with uptake of drugs , even though most attention so far has been on the former , . thus , it is necessary to look at other characteristics of the health system to explain why some european citizens wait longer to receive innovative treatments than others , , especially given the success of harmonization of the registration process . of the more recently introduced pharmaceuticals ; oxalaplatin was first approved in france in 1996 , and approved throughout the eu through the mutual recognition procedure in 1999 , france being the reference member state . zidovudine was first approved in 1987 using national licenses , before the concertation procedure became mandatory for hiv / aids treatments . for these pharmaceuticals , the variation in timing in introduction was low among the countries , including estonia . for tamoxifen , there was a time lag in several countries between the registration of the drug and its use in clinical trials . at that time , several therapeutical strategies were being discussed and its role of tamoxifen seems not to have been clearly defined . scientific papers at that time argue that it had a limited role and called for more clinical trials , which started in most of the countries in this study in the late 1970s and 1980s . to define the start of the diffusion process , one needs empirical longitudinal data on the uptake of drugs , such as sales statistics . however , in most cases , these were only available for recent years when the drugs were already registered for some years . thus , sales statistics were mainly used to verify that the drug had diffused . instead , diffusion was often considered to have begun with the first clinical trials in each country , provided indicators of a continued diffusion were available . for tamoxifen and antiretroviral drugs , there may also have been less well documented use of the drug in some countries not captured by the search for clinical trials so the variation between countries should therefore be interpreted with caution . there were several indications of a continued diffusion of the different innovations . for several innovations , the length of period of diffusion seems to be similar among the countries according to sales statistics . for these pharmaceuticals , variations in use may instead reflect variation in the start of the diffusion among the countries . in most cases , there was a time lag of between 7 and 17 years from the introduction until guidelines were published . thus , the presence of clinical guidelines would not reflect an early phase of the diffusion of innovation promoted by innovators and early adaptors . a previous review have found that the mean compliance rate for guidelines was 54% 3 years after the publication . thus , it seems as if the publication of guidelines may reflect the phase when the innovation diffuses from the early majority to the late majority . this would be in line with the theory of diffusion of innovation , where the late majority is considered to be more conservative and will not adopt an innovation until it is standard practice . several modifiable reasons have been found for not using proven pharmaceutical interventions , including clinician , patient and system factors . thus , it may be possible to influence the implementation of a pharmaceutical innovation in order to limit the variation in introduction and diffusion . in order to establish a new medical drug in medical practice , its efficacy must be proved in clinical trials and the drug must be approved by the regulatory authorities based on data on efficacy and safety . efficacy refers to the potential of improving health of the patients under ideal conditions of use , which should be the case in clinical trials . the pharmaceuticals studied in this paper have all passed this phase of assessment and have been approved for use in medical practice . however , we must also consider that the effectiveness , defined as what is achieved under ordinary conditions , may vary . there may be variation in the uptake of pharmaceuticals and other medical interventions and the quality of care may differ . thus , the outcome of medical care may differ from what would be expected from the results of clinical trials . as pointed out by brook and lohr , there is need to integrate the concepts of efficacy , effectiveness , variations in population - based rates of use and quality of care when building models for policy , planning and evaluation of medical care . in order to assess the impact on populations health of a new medical drug , we have to be able to measure the timing of introduction of the new drug as well as the diffusion process . this information should then be linked to population outcome measures , in order to assess whether the new treatment has been effective in improving the health of the population . although the introduction of a new drug may be regarded as the date of registration , this paper has shown that the year when the drug was first used in medical care often differs from the official registration year . according to the theory of diffusion of innovations , the medical intervention may be introduced by innovators but in order to have a broader impact on populations health , there must also be a continued diffusion of the innovation . when applying this concept , we found that the registration year of the pharmaceuticals studied may differ from the year of introduction with a range between 11 years before and after the registration year . in assessing any impact on population health , therefore , the key issue is when the drug came into widespread use . however , although we used a wide range of data in order to ascertain the introduction and diffusion of new innovations , our main finding is the need for harmonized data in eu countries . the lack of data on different phases of the diffusion process limited the possibilities to analyse the impact of introduction and diffusion of the new drugs . data on the diffusion of drugs and not simply the registration year should routinely be documented in a comparable way across the eu . this is most likely going to require prescribing data , which at present is inconsistent and only some elements are in the public domain . this should , ideally , include information on the indications for prescribing , although clearly , this will not be possible for some time , except where functioning patient information systems at a population level exist . it will also be important to use harmonized measures of the amount of drugs prescribed , such as the ddd . such improvements in the information on the introduction and diffusion of drugs would be a valuable tool in evaluating and planning health care . several pharmaceuticals first available in the 1970s and 1980s were introduced fairly simultaneously in most countries with the exception for soviet era estonia , in which the introduction was delayed until the 1990s . the delay in introducing the pharmaceuticals was shortest in france , the netherlands and united kingdom . this was true both for year of registration and first indicators of diffusion of the drugs , suggesting that delays are not only due to differences in the processes of registration . however , comparisons are challenging and there is a need for improved information on the introduction and diffusion of therapeutic innovations to inform comparisons of health system performance .

focal segmental glomerulosclerosis ( fsgs ) causes glomerular lesions that can progress to end - stage renal disease . it is suspected to be caused by a circulating factor that is amenable to plasmapheresis removal and exhibits a risk for recurrence in the renal allograft . we present two patients with fsgs recurrence in their allograft kidneys diagnosed by biopsy after significant proteinuria developed in the posttransplant setting . treatment with therapeutic plasma exchange induced long - term remission in both patients . spot urine protein : creatinine ratios were monitored and treatment was continued until a target of < 0.5 was achieved . in patient number two , a second peak in proteinuria and azotemia was ultimately attributable to ureteral stenosis and these values normalized following repair . in conclusion , therapeutic plasma exchange is an effective treatment for fsgs recurring following renal transplant . rationale , aims and objectivesdifferences in the performance of medical care may be due to variation in the introduction and diffusion of medical innovations . the objective of this paper is to compare seven european countries ( united kingdom , the netherlands , west germany , france , spain , estonia and sweden ) with regard to the year of introduction of six specific pharmaceutical innovations ( antiretroviral drugs , cimetidine , tamoxifen , cisplatin , oxalaplatin and cyclosporin ) that may have had important population health impacts.methodswe collected information on introduction and further diffusion of drugs using searches in the national and international literature , and questionnaires to national informants . we combined various sources of information , both official years of registration and other indicators of introduction ( clinical trials , guidelines , evaluation reports , sales statistics).results and conclusionsthe total length of the period between first and last introduction varied between 8 years for antiretroviral drugs and 22 years for cisplatin . introduction in estonia was generally delayed until the 1990s . the average time lags were smallest in france ( 2.2 years ) , united kingdom ( 2.8 years ) and the netherlands ( 3.5 years ) . similar rank orders were seen for year of registration suggesting that introduction lags are not only explained by differences in the process of registration . we discuss possible reasons for these between - country differences and implications for the evaluation of medical care .

breast cancer , first described as early as 3000 b.c . by edwin smith papyrus of egypt , comprises 23% of all female malignancies ( excluding non melanomatous skin cancer ) [ 1 , 2 ] . in 2008 , the number of deaths from breast cancer totaled 460,000 patients ; it is a global concern accounting for 14% of all cancer deaths in females . it is the most common invasive cancer in women with an incidence that ranges between 19.3 per 100,000 in eastern africa to 89.7 per 100,000 in western europe [ 4 , 5 ] . the mortality in breast cancer patients is attributed to metastatic disease [ 3 , 6 ] . it is known to metastasize to numerous organs including lymph nodes , lung , bone , liver , skin , kidneys , brain , adrenal , thyroid , and heart [ 3 , 6 , 7 , 8 ] . breast cancer metastasizing to the urinary bladder has only been reported sporadically totaling 41 cases in the english medical literature [ 3 , 913 ] . bladder metastasis from breast cancer as the only organ involved is very rare , with only eight cases reported worldwide [ 3 , 11 , 12 , 14 ] . we herein present a patient who presented with bladder metastasis from breast cancer with the bladder being the only organ involved . a 64year old female patient , a non smoker known to have hypertension , diabetes mellitus , dyslipidemia , supraventricular tachycardia , and osteoporosis was diagnosed in 2005 with left breast intraductal carcinoma . ten out of 23 nodes were positive ; she was staged as t2 ( 4 cm ) n3 m0 disease . the patient was treated with eight cycles of chemotherapy , adriamycin , cytoxan , and taxol and 25 sessions of radiation therapy ( total dose of 50 grays ) , and completed a 5year treatment with an aromatase inhibitor . she had no evidence of disease until march 2010 , when she presented to our clinic with recurrent urinary tract infections and urinary incontinence that failed to resolve with antibiotics alone . workup included a computed tomography ( ct ) scan of her abdomen and pelvis , which showed diffuse thickening of the urinary bladder wall with surrounding fat streaking of the pelvis and retroperitoneum ( figure 1 ) . there was evidence of mild right hydroureteronephrosis with no evidence of obstructing urinary tract calculi ( figure 1 ) . a , b. diffuse thickening of the urinary bladder wall ( arrows ) with surrounding fat streaking of the pelvis . a pocket of gas is noted c , d. mild right hydroureteronephrosis ( arrowheads ) . pathology showed an unremarkable bladder mucosa , but a submucosal nest of carcinoma cells was found . the cells were plasmacytoid with marked nuclear pleomorphism , frequent mitotic figures and multiple foci suggestive of lymphovascular invasion were present . the tumor cells were positive for estrogen receptor and e cadherin ; they were negative for progesterone receptors . a. h&e sections of the urinary bladder biopsy revealing a dense submucosal infiltrate ( mag . b. the cells are cohesive , plasmacytoid with an abundant eosinophilic cytoplasm and eccentric nucleus ( mag . the cells demonstrate positive immunostaining with anti estrogen receptor ( er ) antibody ( c , mag . chest ct scan and bone scan were performed as part of the full work up and failed to show any evidence of other distant metastasis . one week after receiving the first cycle of chemotherapy , the patient developed hematuria and clot retention . several attempts of irrigation failed so the decision was made to perform cystoscopy in order to fulgurate all bleeders . the cystoscopy identified a large clot in the bladder , which was removed and all bleeding areas were fulgurated . . however , a few days after the third chemotherapy cycle she developed severe dyspnea and was found to have pneumonia that progressed to septic shock . the patient passed away from cardiorespiratory arrest one year after the diagnosis of the bladder metastasis . until 2012 , 41 cases of bladder metastasis from breast cancer have been reported and they were mostly associated with systemic dissemination and multiple organ involvement . only eight cases of solitary bladder metastasis from primary breast cancer have been documented and our case represents the ninth in the english medical literature [ 3 , 11 , 12 , 14 ] ( table i , in bold ) . macroscopically , bladder metastasis may appear as a mass , irregular lesion , mucosal nodularity , abnormally thickened bladder wall , or plaque with telangiectasias . cystoscopy was performed based on the suspicious ct scan findings , and it revealed an abnormal bladder wall thickening and inflammation , which were both subsequently biopsied . abbreviations : gu genitourinary , nr not reported , mets metastasis , ln lymph node , sb small bowels , lb large bowels , ilc invasive lobular carcinoma , idc invasive ductal carcinoma , er estrogene receptors , pr progesterone receptors the breast primary tumor subtype was invasive intraductal carcinoma while in the previously published reports , the most common histology of the breast primary was invasive lobular ( 10 out of the 15 cases where the breast cancer subtype was determined ) . bates and baithun reported 4.5% incidence of secondary bladder metastasis among all bladder cancer [ 9 , 15 ] , with secondary metastasis to the bladder from breast cancer being approximately 3% [ 16 , 17 ] . when autopsy and pathology are used as mainstay for diagnosis , the incidence ranges from 0% to 7% . bladder metastasis from previously diagnosed breast cancer is reported in the literature to vary from 2% to 14% . the most common primary tumors metastasizing to the bladder are : stomach , lung , skin / melanoma , and breast . it is only when the mucosa is involved by the disease will symptoms become clinically detectable . since mucosal involvement is the last stage of metastatic invasiveness into the bladder , the prognosis is very poor with a mean survival of two to three years , although a 5year survival of two patients out of the 41 was reported in the literature [ 14 , 20 ] . as a consequence , early stages of breast cancer metastasizing to the bladder the most common findings in patients with secondary bladder metastasis are lower urinary tract symptoms ( luts ) , flank or abdominal pain , hydronephrosis , and the painless hematuria that is in many cases the most common initial symptom ( microscopic being more frequent than gross ) . hematuria as a sign of bladder involvement following primary breast cancer is considered sensitive , but not specific for tumor metastasis . gross hematuria with a history of breast cancer needs to be thoroughly investigated , keeping in mind the side effects of cyclophosphamide as treatment of the primary breast cancer , regardless of time or duration of treatment . suprapubic and bilateral flank pain was later the major disabling symptom that warranted the investigation through ct scan imaging . ca 153 is one method to follow up breast cancer recurrence or metastasis , but strong evidence are lacking on its clinical usefulness . the positron emission tomography ( pet ) scan has been showed to have increasing usage after suspecting bladder involvement in a breast cancer patient ; however , its cost effectiveness is yet to be determined . breast metastasis to the bladder has been shown to have a worse prognosis than metastasis to bone . the time interval between primary tumor diagnosis and detection of metastasis is highly variable between 0 month and 30 years with an average of 6.2 years ( table 1 ) . bladder metastases in our patient were identified five years after the initial diagnosis of primary breast carcinoma . conduction of the proper investigations prevented the delay in the diagnosis of the metastatic disease . the patient survived one year from the time she first presented with urinary symptoms , and there was no evidence that her death was related to the bladder metastasis . only 8% of all breast cancer is lobular carcinoma , however , it is the most common type of breast cancer type involving the bladder ( 33% of secondary bladder metastasis ) followed by ductal carcinoma , which accounts for the majority of primary breast cancer ( 66% ) and metastasizes mostly to the lung parenchyma . one hypothesis is that lobular carcinoma is of the serosal type , which gives it a predilection to spread to the gastrointestinal and gynecological systems . it is part of the seeding soil hypothesis : the interaction of tumor with specific host factors in the metastasized organ . another culprit is concomitant steroid therapy , which is thought to be due to exacerbation of the immunosuppressive effect . estrogen , progesterone , and her2 receptors are the three main receptors studied in breast cancer . discrepancy between receptors is not uncommon between the primary and the secondary tumor ( reported between 30 and 39% ) . bladder metastases from our case were positive for estrogen receptors , which was also true for the patient 's known primary cancer of the breast . however , progesterone receptors were only present in the malignant breast cells and not the secondary bladder metastasis . one hypothesis is that the polyclonicity of breast tumor cells is affected by treatment modalities ( hormonal therapy may select some and suppress others ) , which manifests itself later in case of bladder metastasis . it has been shown that if receptors convert from positive in the breast to negative in the bladder , it is associated with decrease survival [ 17 , 24 ] . even with receptor negative secondary bladder metastasis , a trial of anti receptor therapy has been used with promising results in controlling disease . reported cases of bladder metastasis were managed through surgery , chemotherapy , radiotherapy , hormonal therapy , or a combination of those . in our patient , we report a rare case of breast cancer with solitary urinary bladder metastasis that was diagnosed several years after the initial presentation . secondary malignancies of the bladder are difficult to distinguish from non transitional cell primary bladder cancer . a high level of suspicion and extensive investigation are warranted if a known primary cancer already exists . we emphasize the need to be more aware of the possible metastatic nature of every urinary symptom that shows in a breast cancer patient . coronary artery fistulae are direct connections from one or more coronary arteries to cardiac chambers or a large vessel . congenital anomalies are more common than acquired,1)2 ) being found in up to 0.2% of routine angiocardiography.3 ) most reported cases are about coronary artery fistulae to right cardiac structure,4 ) but the fistula between coronary artery and left ventricle is considerably rare.4 - 6 ) moreover , a case of multiple fistula emptied into the left ventricle through the entire left ventricular walls including left ventricular free wall , interventricular septum and apex is uncommon . the following we presented is such a case , with impressive echocardiac images which demonstrating diastolic massive abnormal color doppler flows on the entire left ventricular walls which were mimicking firecracker on the whole left ventricle . a 31-year - old woman was referred from local clinic for further investigation of cardiac murmur . past health status was good . physical examination revealed a blood pressure of 107/43 mmhg , and regular pulse of 86 beats / min . electrocardiography demonstrated biphasic t wave in lead v1 - 3 and borderline prolongation of qt interval ( fig . transthoracic echocardiography demonstrated a normal functioning left ventricle with hypertrophy and trabeculation of left ventricular free wall and interventricular septum ( fig . 2 ) . imaging with color flow doppler demonstrated multiple sparkling colors doppler flow through left ventricular free wall and interventricular septum during diastole ( using a 4 - 6 mhz - frequency transducer and high nyquist limit : 74 ) , and these multiple sparkling color flows demonstrated multiple fistula formation from the epicardial surface into the entire left ventricular cavity through the hypertrophied segment of myocardium during diastole ( fig . it also showed suspicious non - compacting myocardium at apical infero - posterior wall of left ventricle . we observed two separated layers of myocardium with a thin , compacted , epicardial layer and a thick , non - compacted endocardial zone . the ratio of non - compacted myocardium to compact myocardium at the end of systole was 2.9 : 1 with mean non - compacted myocardium of 1.3 cm and compacted myocardium thickness mean of 0.4 cm . for detailed anatomical information , the computerized tomographic ( ct ) angiography also identified multiple fistula from the left coronary artery towards the entire left ventricular walls including left ventricular free wall , interventricular septum and apex . coronary artery had markedly tortuous and dilated morphology from left main coronary artery to left anterior descending artery . coronary artery fistulae are rare and are most often diagnosed by angiography incidentally . in our case , because of the prominent firecracker on the whole left ventricle , we could make the early detection of coronary fistulae by noninvasive transthoracic echocardiography without invasive coronary angiography . imaging with color flow doppler , using a 4 - 7 mhz - frequency transducer and high nyquist limit revealed unusual multiple high velocity color - flows from the epicardial surface into the left ventricular cavity during diastole . it seemed that those multiple diastolic mosaic flows reflect the presence of fistula between epicardial coronary system and left ventricular cavity . left ventricular walls including left ventricular free wall and interventricular septum had sinusoidal appearance and mesh like structure which were plexus of multiple small vessels emptying exclusively into the left ventricle through the whole left ventricular wall , and also she had marked trabeculations and deep sinusoidal recesses involving the apex , apico - lateral and apico - inferior walls . this exam revealed two separated layers of myocardium with the ratio of non - compacted to compacted myocardium was greater than 2 , measured at end systole in the parasternal short - axis view . the definition and diagnostic criteria of non - compaction is not yet fully established , but non - compaction is generally characterized by continuity between the ventricular cavity and the intertrabecular recesses , which are filled with blood from the ventricle and which have no communication with the epicardial coronary system . however , in the presented case , which had multiple direct communications with the epicardial coronary system through the entire left ventricular wall . in that point , we concluded this case is more compatible with multiple fistula than non - compaction . although the etiology of non - compaction has not been fully investigated , our case is thought to result from an arrest in the regression of embryonic sinusoids during the embryonic period . the arrest of myocardial compaction that occurs in the early stages of fetal development result in the persistence of intertrabecular spaces and incomplete formation of capillaries . in this case , we believed that this process have a role in the formation of multiple fistula with the ventricular cavity and epicardial coronary circulation . the main limitation of our case was that we could not perform angiography to confirm coronary artery fistula . but ct angiographic finding was also compatible with multiple fistula draining into the left ventricle . in the presented case , the left ventricular functions were preserved , although the fistulae were very extensive . in a patient with coronary artery fistulae , myocardial ischemia and diastolic volume overload of the left ventricle can be caused by left - to - left shunt : however , the clinical and hemodynamic consequences are incompletely understood . in the presented case , the patient had no symptom and refused further evaluations including coronary angiography and catheterization . with regard to this special case , due to the fact of patient 's refusal and absence of angina , it was decided clinical follow - up of the patient . she remained asymptomatic and no complicate events were recorded in the next 5-year follow - up . in conclusion , this case report represents multiple fistula emptied into the left ventricle through the entire left ventricular walls including left ventricular free wall , interventricular septum and apex , confirmed by transthoracic echocardiography and ct angiography .

breast cancer is the most common malignancy in woman . the urinary bladder is an unusual site for metastasis from primary tumors of the breast , especially when it is the only organ involved . we present the case of a female patient with known breast cancer stage t2n3m0 who developed isolated bladder metastasis five years after the primary diagnosis . we reviewed the literature for similar cases and discussed the clinical presentation , pathophysiology , and prognosis of this entity . coronary artery fistulae are usually identified during invasive coronary angiographies . however , in this case , we made the early detection of coronary artery fistulae during non - invasive transthoracic echocardiography , by demonstrating diastolic multiple abnormal color doppler flows on the entire left ventricular walls including left ventricular free wall , interventricular septum and apex , which were mimicking firecracker on the whole left ventricle . fistulous communication from the coronary artery to the left ventricle is rare . moreover , a case of multiple coronary fistulae emptying into the left ventricle through the entire left ventricular walls including left ventricular free wall , interventricular septum and apex is uncommon . we report a case of a 31-year - old woman who was diagnosed with multiple fistula communicating with entire left ventricular wall .

the detection and quantification of tumour - specific rearrangements are important issues in cancer research and in clinical diagnosis of tumours . in particular , its significance became obvious for haematological malignancies that exhibit characteristic translocations in specific tumour subgroups . although gene rearrangements are typical for haematological malignancies , they also may occur in solid tumours as characteristic changes . this has been shown for ret / ptc rearrangements in papillary thyroid carcinoma ( ptc ) that fuse the ret proto - oncogene to a variety of constitutively expressed partner genes ( for review see zitzelsberger ) . this was further improved by the development of fluorescence in situ hybridization ( fish ) techniques that allows a cytogenetic analysis of rearrangements on metaphase spreads as well as on interphase cell nuclei . multicolour fish approaches such as spectral karyotyping ( sky ) allowed a more detailed analysis of cytogenetic aberrations , in particular in the case of complex and hidden rearrangements [ 5 , 6 ] . the analysis of interphase nuclei by fish has the advantage that gene rearrangements can be investigated at single cell level in nonproliferating cells . an evaluation of fish signals is usually performed by visual inspection directly from the microscopic image . in this case , cell numbers for further statistical analysis and a possible bias of the investigator towards positivity or negativity of fish signals indicating the rearrangement are major limitations . in order to analyse a statistically relevant number of cells , an automatic scanning system for fluorescence spot counting using a fully motorized fluorescence microscope with an eight - slide scanning stage and a high - resolution ccd camera driven by the metacyte software ( metasystems , altlussheim , germany ) has been established and optimized . to demonstrate routine application of the scanning system , the ret / ptc rearrangement in papillary thyroid carcinomas has been scored with a probe set that produces split fish signals if a gene rearrangement is present . therefore , the parameters of the scanning system had to be optimized using cell culture models as positive and negative controls . the aims of the present study were to establish such optimised scanning parameters and to characterise chromosomal and ret / ptc rearrangements in a ptc cohort . primary cell cultures of 23 ptcs from children and adults from ukraine that developed papillary thyroid carcinomas in the aftermath of the chernobyl accident were established according to a published protocol . the median age of the patients at operation was 21 years , ten patients were male , and 13 patients were female . 21 out of 23 cases were investigated for chromosomal aberrations and 22 cases for ret / ptc rearrangements . in addition , a cell line originating from a ptc ( tpc1 ) carrying the ret / ptc1 rearrangement served as a positive control [ 9 , 10 ] . as negative control we used a cell line derived from human retinal epithelium ( rpe , htert immortalised ) that displays a normal karyotype . all cell lines and primary cell cultures were grown in rpmi 1640 ( paa laboratories , clbe , germany ) with the addition of penicillin ( 5 iu / ml ) and streptomycin ( 5 g / ml ) ( gibco - brl life technologies , karlsruhe , germany ) and supplemented with 10% or 20% fbs ( sigma , taufkirchen , germany ) , respectively . therefore 2.5 10 cells were grown in 4 ml media on a sterile glass slide positioned in quadriperm cell culture chambers ( in vitro systems and services gmbh , gttingen , germany ) and addition of 0.05 g / ml colcemid ( roche , penzberg , germany ) overnight arrested cells in metaphase . after 2432 h growth , the media were removed and the slide covered with 4 ml hypotonic kcl - solution ( 0.075 m ) . after incubation under hypotonic conditions for 20 min at 37c , 4 ml of ice - cold fixative ( methanol / glacial acetic acid , 3 : 1 ) were added followed by another incubation step for 20 min on ice . subsequently the solutions were removed and replaced by another 4 ml of ice - cold fixative . finally , the slides were air - dried perpendicularly under a laminar flow . for interphase preparations , cells cells were fixed with carnoy 's fixative ( methanol / acetic acid ; 3 : 1 ) , air - dried , and stored at room temperature for 7 days before hybridization . briefly , metaphase preparations were pretreated with rnase a ( 0.1 mg / ml in 2 ssc ) prior to hybridization . chromosome denaturation was achieved by treatment of the slides in 70% formamide in 2 ssc at 72c for 1 - 2 min . subsequently the slides were dehydrated in a 70% , 90% , and 100% ethanol series and hybridized with a denatured sky - probe mixture ( skypaint dna kit , applied spectral imaging , mannheim , germany ) . after hybridization ( 24 h ) , slides were washed in 0.5 ssc for 5 min at 75c , 4 ssc/0.1% tween for 2 min and h2obidest for 2 min , both at room temperature . probe detection was achieved using antidigoxigenin ( 1 : 250 ; roche , penzberg , germany ) , avidin - cy-5 , and avidin - cy-5.5 antibodies ( both 1 : 100 ; biomol , hamburg , germany ) according to the manufacturers ' protocols . metaphase spreads were counterstained with 0.1% 4,6-diamidino-2-phenylindole ( dapi ) in antifade solution ( vectashield mounting medium ; vector laboratories , burlingame , ca , usa ) . a minimum of 15 metaphases were analyzed to determine the karyotype of each primary culture . image acquisition was done using a spectracube system , and analyses were accomplished using the skyview imaging software ( both from applied spectral imaging , mannheim , germany ) . for fish analysis of ret / ptc rearrangements , labelling of yac dna probes 344h4 , 214h10 and , the yac probes 313f4 and 214h10 map proximal to and include the ret locus , whilst clone 344h4 contains dna sequences distal to ret . they were labelled either with digoxigenin-11-dutp ( 344h4 ) or with biotin-16-dutp ( 214h10 , 313f4 ) using nick translation and were detected with antidigoxigenin - cy3 antibody , followed by rat - anti - mouse cy3 and mouse - anti - rat cy3 for red fluorescence , and streptavidin - fitc , followed by biotin - antistreptavidin and streptavidin - fitc , respectively . a normal ret locus results into two overlapping red and green fish signals , while split fish signals ( separated red and green signals ) indicated a rearranged ret gene . only cells with either two overlapping signals or one split and one overlapping signal were analysed to ensure completeness of the nuclei . for the analysis of the ret / ptc rearrangements , a fluorescence - based scanning system , metafer4 ( metasystems , altlussheim , germany ) , was used . this scanning system is based on a motorized axioplan 2 microscope ( zeiss , oberkochen , germany ) , a motorized eight - slide scanning stage ( mrzhuser , wetzlar , germany ) , and high - resolution ccd camera ( jai corporation , japan ) . the classifiers of the metacyte software allow the settings of image capture , exposure parameters , image processing , and cell processing steps . these classifiers contain variable criteria for cell selection taking into account cell characteristics like cell area , aspect ratio , concavity index , and signal intensity . for capturing of cell images , slide scanning procedure was started with the focus determination of cells in the dapi channel in the selected scanning area . then , a stack of five images within a distance 0.9 m were captured in the cy3 and fitc channels . from each stack , a 2d image with all signals in focus was created and a local background reduction was performed applying two standard top hat filters . overlapping or incomplete cell nuclei as well as nuclei with incorrect numbers of red and green signals were excluded from the analysis . for the analysis of fused or split signals , the distance of signals was measured in the xy positions as well as in z positions . between 100 and 870 cells each 2d image was displayed as gallery pictures presenting the cell number , the number of red and green signals , and the number of overlapping red and green signals . on the gallery screen also the results for each sample could be displayed as scatter diagram and/or as bar diagram . all data can be exported into common statistics and graphic software programs . in parallel , every captured cell was analysed visually in order to compare visual and automated scoring of fish signals . in contrast to automated scoring the visual analysis can be performed in two dimensions only . for optimization of the classifier parameters for ret / ptc rearrangements exhibiting split signals negative and positive control samples were analysed several times after changing the parameter settings for image capture , cell nucleus characteristics , and cell nucleus selection , by criteria such as nuclear area , aspect ratio , and concavity index of the nuclei , as well as size and distance of red and green signals . the final parameter settings for the automated analysis were optimised for the lowest numbers of false positive and false negative results . frequencies of cells with split signals determined by automated analyses and by visual analyses were compared with an test . significant differences were accepted for p values less than .05 after adjustment for false detection by the bonferroni method . 21 ptcs were analyzed for chromosomal aberrations , and 22 ptcs were investigated for ret / ptc rearrangements . the ret / ptc status was determined by fish analysis on interphase cells from the same primary cultures that were used for sky analysis of chromosomal aberrations . for an evaluation of ret / ptc rearrangements an automated scoring system ( metafer4 ) was used and data were compared to fish signals that had been scored visually . sky analysis revealed clonal chromosomal rearrangements in five out of 21 cases ( 24% ) . in two cases ( 10% ) structural aberrations involving chromosome 11 could be observed . additionally , numerical chromosome aberrations could be detected in 13 cases ( 62% ) with frequent losses of chromosomes 21 ( six cases , 29% ) , 20 ( five cases , 24% ) , 7 , 10 , and 22 ( three cases each , 14% ) . figure 1 shows an exemplary sky image of case 402 t exhibiting a deletion on chromosome 11 and i(11)(q10 ) . fish was carried out on interphase cell preparations from primary cultures of 22 ptcs using a combination of three yac probes that were labelled in two different colours . cell nuclei exhibiting a rearranged ret gene show a split fish signal in red and green in addition to an overlapping fish signal , whereas normal cells show two overlapping fish signals ( figure 2 ) . sixteen out of 22 ptcs ( 72% ) exhibited ret rearrangements diagnosed by fish interphase analysis ( table 1 ) . the highest frequency of rearranged cells after fish interphase analysis was 41% ( case s430 t ) . these 16 cases showed a significantly elevated frequency of split fish signals compared to rpe cell line and s414 normal tissue which represent negative controls without any ret / ptc rearrangement . the frequency of false positive fish signals in these control cells is in the order of 13% ( table 1 ) . therefore , we used a threshold of 7.1% of cells with a split fish signal in order to define a ret / ptc - positive tumour as published earlier in . the positive control cell line tpc1 showed 98% rearranged cells . in order to create the initial parameter settings for classifiers of the automated metafer4 scoring system the rpe ( human retina pigment epithelial ) cell line and primary culture of normal tissue from case s414 , both with a normal karyotype , were used as negative control . the tpc1 cell line that was derived from a human papillary carcinoma carrying a ret / ptc1 rearrangement was used as a positive control . based on these control cells the classifiers of the metafer4 system were optimized several times by changing the parameter settings for cell nucleus characteristics and cell nucleus selection by criteria such as nuclear area , aspect ratio , and concavity index of the nuclei . for the scoring of fused and split fish signals an optimisation of the parameters for minimum signal area , maximum distance of red and green signals , and the minimum signal intensity has been performed in multiple training procedures . for testing the final parameter settings of the classifier the above - mentioned 22 ptcs were analysed using the automated scoring system as well as visual scoring of the recorded images . the negative control cell lines showed a frequency of false positive ret / ptc rearrangement of 13% . the frequencies of ret / ptc positive cells among the ptc primary cultures varied between 1.0% and 41.5% . concordance of results from automated and visual scoring was tested statistically . statistical correlation analysis using the test confirmed a concordance of the data from automated and visual analysis in 19 cases ( 86% ) . only in three cases ( s407 t , s418 t , and s422 t ) the automated and visual analysis differed significantly ( p < .05 ; bonferroni value , figure 3 ) . in this study , we describe karyotype abnormalities and rearrangements of the ret proto - oncogene in ptc from patients that were exposed to the radioiodine fallout of the chernobyl accident . there is evidence from several studies that exposure to ionising radiation leads to the induction of chromosomal rearrangements that may result in gene alterations and deregulated gene expression [ 13 , 14 ] . in ptc chromosomal breakpoints on 1p32 - 36 , 1p11 - 13 , 3p25 - 26 , and 7q32 - 36 have previously been reported after conventional karyotyping . other cytogenetic studies on ptc showed a deletion on chromosome 11q and a chromosome 2 rearrangement with an assumed tyrosine kinase gene at the breakpoint . in addition , novel breakpoints of structural rearrangements of chromosomes 4q , 5q , 6p , 12q , 13q , and 14q and of complex rearrangements have been reported using a complementary analysis of conventional karyotyping , sky and fish with bac clones [ 8 , 18 , 19 ] . in our study , also rearrangements involving chromosome 11 could be detected in two cases ( table 1 , figure 1 ) , indicating an important gene on this chromosome that might be involved in thyroid carcinogenesis . besides the structural rearrangements it is remarkable that many numerical aberrations were observed mostly involving chromosomes 20 , 21 , and 22 . loss of chromosome 22 has already been reported to represent a cytogenetic marker for poor prognosis in thyroid cancer [ 2022 ] . thus , this observation in ptc may explain to some extent the more aggressive phenotypes of tumours that have developed after the chernobyl accident . another frequent cytogenetic finding in ptc is the rearrangements of chromosome 10q with breakpoints at 10q11.2 that lead to an activation of the ret proto - oncogene [ 23 , 24 ] . the most frequent rearrangements are paracentric inversions on chromosome 10q leading to the oncogenes ret / ptc1 and ret / ptc3 . thus , these chromosomal rearrangements lead to transcribed fusion genes that affect the mapkinase pathway . investigations of these ret / ptc rearrangements are important in ptc since they represent frequent alterations and molecular targets for therapeutic interventions [ 25 , 26 ] . in this study , we have investigated ret / ptc rearrangements in the ptc using an interphase fish approach that allows to detect ret rearrangements regardless of the specific fusion partner involved at a single - cell level . the frequency of ret / ptc - positive cases of 72% is in line with earlier reports on tissue sections using a three - dimensional evaluation of fish signals either with laser - scanning microscopy or apotome - equipped fluorescence microscopy [ 7 , 27 ] . the reported genetic heterogeneity could be confirmed in this study since only a subpopulation of tumour cells showed the ret / ptc rearrangement . although interphase fish analysis has a number of advantages in detecting gene rearrangements , it is a challenge to score adequately the frequency of rearranged cells . a major problem that also affects the sensitivity of the fish approach is the inevitable scoring of false positive fish signals due to random generation of rearrangement - positive fish signals . the choice of adequate fish probes ( fusion probes or split probes for the detection of a rearranged cell ) is one issue that has to be addressed ; an impartial evaluation of fish signals is another important issue . in this study , we have compared visual inspection of fish signals with an automated evaluation . in the automated process , a correct detection of fish signals is a great challenge , since fish signals exhibit large variations in shape , size , and intensity . after cell nucleus selection and correct fish signal detection , a discrimination of nuclei with and without ret / ptc rearrangement could be achieved by measuring the signal distances and defining the minimal distance to diagnose a split fish signal . tpc1 cell line has this feature and showed in our study 97% and 98% positivity , respectively . the automated analysis reported in this study was optimised to detect ret / ptc rearrangements by means of a yac probe set covering the ret gene locus . differential labelling of the yac probes in red and green resulted in overlapping red / green signals for the wild - type ret gene and in a split red and green fish signals for the rearrangement . the automated evaluation of fish signals revealed matching results in 87% of the 22 cases with the visual analysis of fish signals . a possible explanation for three misclassified cases is a bias of the investigator scoring visually towards ret / ptc - positive or -negative cells , especially in cases with high background signals . also different cell features in those three cases an additional difference in fusion counting automatically or by eye is that the automated system is measuring the distance 3-dimensionally , whereas directly at the microscope or with captured images a visual scoring is performed at 2d projection . a comparison of automated and manual evaluation of interphase fish results was only presented by kajtr et al in case of bcr / abl rearrangements in cml patients . although the probe design is different ( fusion probe versus split signal probe ) , we received a similar good concordance of automated and manual evaluation results . we have demonstrated here for the first time the usage of the 3d automated fish analysis for the detection of ret / ptc rearrangements in ptc . in conclusion , we have shown that chromosomal rearrangements ( 5 out of 21 ) and rearrangements of the ret gene ( 16 out of 22 ) are frequent in papillary thyroid carcinomas from patients of the ukraine after the chernobyl accident . for the detection of ret / ptc rearrangements we have demonstrated an automated fish analysis approach which provides reliable results in higher cell numbers . the results of ret / ptc rearrangements again indicate a genetic heterogeneity since only subpopulations of tumour cells carried the ret / ptc rearrangement . hurling is a stick and ball invasion field sport native to ireland ( reilly and collins , 2008 ) . the movement profile of players is stochastic in nature , where the timing of high speed running follows the ebb and flow of competitive play ( reilly and collins , 2008 ) . despite its spectator popularity very few research investigations have been conducted on hurling cohorts ( reilly and collins , 2008 ) . the training - recovery relationship is an important consideration in the training prescription of players and a clear understanding of training loads can help inform practice . recent reports have linked the training load and inadequate fitness to the high number of injuries observed within the gaelic sports ( blake et al . , 2014 ) . ( 2014 ) reported that the injury incidence rate for match play ( 61.75/1000 hr ) is 21 times that of training ( 2.99/1000 hr ) . this suggests that training does not adequately prepare players for the competitive demands of match - play . the training load ( tl ) in field sports can be divided into internal ( heart rate , rpe ) or external ( distance covered ) responses . an understanding of the tl allows for a more individualised understanding of the dose - response relationship of training and match play ( impellizzeri et al . , 2005 ; stagano et al . , 2007 ) . recent advancement in technology has improved the ease to which locomotion data collection can take place . there has been an increase in monitoring of the external training load in match and training situations through the use of global positioning satellite systems ( gps ) ( casamichana et al . the advent of this technology has resulted in total distance covered and distance covered at arbitrary demarcated thresholds of high speed locomotion being utilised to quantify the tl . the poor sensitivity and variability of these performance metrics within match play and training environments has been previously highlighted ( gregson et al . , 2010 ; kempton et al . , 2014 ; mclaren et al . , 2015 ; stevens et al . , 2016 ) . as a consequence these metrics may be unreliable to gain full appreciation of the tl . the understanding of the dose - response relationship has been advocated as an important consideration for coaches within the training process . monitoring the tl in team sports is difficult due to different exercise designs resulting in different physiological and mechanical demands . additional complications are presented in the form of inter - individual responses to the prescribed exercise ( akubat et al . , 2012 ) . o2 consumption relationship appears to be valid even during intermittent exercise ( esposito et al . , 2004 ) , the itrimp methodology has potential for use for monitoring the dose - response of players to specific training programs within a team sport environment ( manzi et al . , 2013 ) . indeed , measures of the internal tl have been validated with a dose - response relationship in team sports ( akubat et al . , 2012 ; both studies demonstrated that the fully individualized training impulse ( itrimp ) showed relationships with changes in aerobic - fitness measures of soccer players . previous studies ( manzi et al . , 2013 ) have shown that itrimp measures over a training period were correlated to the percentage improvement in vo2max ( r = 0.77 , p = 0.002 ) and yo - yoir1 performance ( r = 0.69 , p = 0.009 ) . ( 2014 ) through the integration of external ( total distance [ td ] ; high speed distance [ hsd ] ) and internal loading parameters ( itrimp ) . interestingly , these measures have shown a relationship with aerobic fitness measures in soccer players ( akubat et al . , 2014 ) . the investigation reported that both td : itrimp showed moderate to large correlations with vobla ( r = 0.65 ; p = 004 ; large ) and vobla ( r = 0.69 ; p = 0.03 ; large ) , with td not significantly related to vlt ( r = 0.28 ; p = 0.43 ; small ) and vobla ( r = 0.14 ; p = 0.69 ; small ) . this highlights the potential merits of using tl ratios to better quantify the dose - response of the tl . furthermore , studies within the rugby league ( weaving et al . , 2014 ) have shown that the integration of tl measures better explain a greater proportion of training component variance that internal or external measures of load alone fail to fully explain . at elite levels of hurling a disproportionate number of injuries occur during match play ( murphy et al . , 2007 ) with most occurring in the last quarter of games due to increased fatigue states ( blake et al . , this is in line with previous investigations ( watson , 1996 ) that have reported injury occurrences in hurling populations which are attributable to poor conditioning of players . given such a scenario , it would appear that a situation where an analysis and assessment of aerobic fitness training through data collected routinely at elite levels could prove useful . considering the potential ease of quantifying the hr ( internal load ) and the gps ( external load ) , both the internal and external output of exercise can be measured . if external : internal measures relate to measures of fitness that predetermine a given performance , more frequent analysis of training status and match performance could be made by practitioners . this would allow for more informed and proactive decisions related to player s fitness and fatigue status , therefore , providing a more thorough understanding of the training load paradigm in intermittent team sports . hence , the current study investigated whether information such as the hr and distance could potentially be used to assess the match play load . therefore , the study aimed to examine a novel approach of integrating external and internal workload variables as a ratio during hurling specific simulated match play . finally , the study aimed to quantify the relationship of integrated tl ratios and aerobic fitness measures in hurling players . twenty - five hurling players ( age : 24 4 yrs ; body height : 1.80 0.02 m ; body mass : 78 3 kg ; vo2max : 57.78 3.05 mlkgmin ) took part in this study . all subjects provided written informed consent to the experimental procedures after all possible benefits and risks were explained to them . ethical consent for the study was granted by the research ethics committee of the institute of technology tallaght . the study conformed to the codes of ethics of the world medical association ( declaration of helsinki , 1964 ) the study was completed over a 3 week period . during the 1 week an incremental vo2max and lactate threshold test ( vo2/lt ) was conducted . the vo2/lt consisted of five ( 8 , 10 , 12 , 14 & 16 kmh ) 4 min stages followed by an increase in speed at a rate of 0.2 kmh every 12 s until exhaustion was reached . the treadmill gradient was set at 1% for the entire test to reflect the energetic cost of outdoor running ( jones and doust , 1996 ) . during the incremental test , each stage was separated by a 1 min recovery period where capillary blood lactate was taken ( lactate plus , nova medical ) in duplicate , with the mean value for each stage recorded . the velocity at 2 mmoll ( vlt ) and velocity at 4 mmoll ( vobla ) were considered as markers of aerobic fitness . maximal aerobic capacity ( vo2max ) was assessed during the vo2/lt through breath by breath analysis ( cosmed k4b , rome , italy ) . vo2max was recorded as the highest mean vo2 obtained for a 1 min period with the following criterion met : ( 1 ) a plateau in vo2 despite increasing treadmill speed ( 2 ) a respiratory ratio above 1.10 ( 3 ) attainment of the age predicted hr ( lucia et al . , 2004 ) . the hr was recorded using hr belts ( polar team system , polar electro , oy , finland ) . the mean hr during the last minute of each stage was utilised to ascertain the individualized hr blood lactate relationship for the calculation of itrimp ( au ) weightings . finally , the peak hr during the incremental treadmill test was considered the peak hr of the participant . during weeks 2 and 3 and at least 5 days after the vo2max / lt test , participants completed a hurling match play simulation protocol . the hurling simulation protocol was chosen given its accurate physiological replication of hurling match play ( collins et al . before conducting the study , test retest reliability analysis was conducted on the hurling match play simulation protocol to ensure that the test provides external validity and reliability . results showed that there was no significant difference between the 1 and 2 trial of the hurling simulation protocol ( 7411 354 m ; 7311 362 m ; p = 0.19 ; trivial ) . the coefficient of variation ( cv ) was 1.9% ( 95% ci : 1.1 - 4.8 ) highlighting the reliability of the simulation . the hurling match play simulation protocol was completed at the same time of the day ( 10.30 14.00 hr ) to account for any circadian variation in performance . during the simulation participants wore an individual gps unit ( vxsport , lower hutt , new zealand , issue : 330a , firmware : 3.26.7.0 ) sampling at 4 hz and containing a triaxial acceloremter and a magnetometer in a total of 30 games . the gps unit ( mass : 76 g ; 48 mm x 20 mm x 87 mm ) was encased within a protective harness between the player s shoulder blades in the upper thoracic - spine region what ensured that players range of movement in the upper limbs and torso was not restricted . the system had been validated , deemed accurate and reliable during intermittent exercise ( buchheit et al . , 2014 ) . following each match gps data were downloaded using the same proprietary software ( vxsport view , new zealand ) . each file was trimmed so that only data recorded when the player was on the field was included for further analysis . the proprietary software provided instantaneous raw velocity data at 0.25 s intervals , which were then exported and placed into a customised microsoft excel spreadsheet ( microsoft , redmond , usa ) . the spreadsheet allowed analysis of distance covered ( m ) in the following categories : total distance ( td ) , high speed distance ( hsd : 17 kmh ) and sprint distance ( sd : 22 kmh ) . internal loading was measured as individualised training impulses ( itrimp ) ( manzi et al . , 2013 ) . pearson correlation coefficients were used to assess the relationships between ( 1 ) aerobic fitness and performance , and ( 2 ) distance : itrimp and fitness . qualitative interpretations of the correlation coefficients and the 95% confidence intervals ( ci ) as defined by hopkins ( 2004 ) ( 0.09 trivial , .10.29 small , .30.49 moderate , .50.69 large , .70.89 very large , .90.99 nearly perfect , 1 perfect ) are provided for all correlations . all statistical analyses were performed using spss for windows ( version 22 , spss inc . chicago , il , usa ) with statistical significance set at an accepted level of p<0.05 . twenty - five hurling players ( age : 24 4 yrs ; body height : 1.80 0.02 m ; body mass : 78 3 kg ; vo2max : 57.78 3.05 mlkgmin ) took part in this study . all subjects provided written informed consent to the experimental procedures after all possible benefits and risks were explained to them . ethical consent for the study was granted by the research ethics committee of the institute of technology tallaght . the study conformed to the codes of ethics of the world medical association ( declaration of helsinki , 1964 ) the vo2/lt consisted of five ( 8 , 10 , 12 , 14 & 16 kmh ) 4 min stages followed by an increase in speed at a rate of 0.2 kmh every 12 s until exhaustion was reached . the treadmill gradient was set at 1% for the entire test to reflect the energetic cost of outdoor running ( jones and doust , 1996 ) . during the incremental test , each stage was separated by a 1 min recovery period where capillary blood lactate was taken ( lactate plus , nova medical ) in duplicate , with the mean value for each stage recorded . the velocity at 2 mmoll ( vlt ) and velocity at 4 mmoll ( vobla ) were considered as markers of aerobic fitness . maximal aerobic capacity ( vo2max ) was assessed during the vo2/lt through breath by breath analysis ( cosmed k4b , rome , italy ) . vo2max was recorded as the highest mean vo2 obtained for a 1 min period with the following criterion met : ( 1 ) a plateau in vo2 despite increasing treadmill speed ( 2 ) a respiratory ratio above 1.10 ( 3 ) attainment of the age predicted hr ( lucia et al . , 2004 ) . the hr was recorded using hr belts ( polar team system , polar electro , oy , finland ) . the mean hr during the last minute of each stage was utilised to ascertain the individualized hr blood lactate relationship for the calculation of itrimp ( au ) weightings . finally , the peak hr during the incremental treadmill test was considered the peak hr of the participant . during weeks 2 and 3 and at least 5 days after the vo2max / lt test , participants completed a hurling match play simulation protocol . the hurling simulation protocol was chosen given its accurate physiological replication of hurling match play ( collins et al . before conducting the study , test retest reliability analysis was conducted on the hurling match play simulation protocol to ensure that the test provides external validity and reliability . results showed that there was no significant difference between the 1 and 2 trial of the hurling simulation protocol ( 7411 354 m ; 7311 362 m ; p = 0.19 ; trivial ) . the coefficient of variation ( cv ) was 1.9% ( 95% ci : 1.1 - 4.8 ) highlighting the reliability of the simulation . the hurling match play simulation protocol was completed at the same time of the day ( 10.30 14.00 hr ) to account for any circadian variation in performance . during the simulation participants wore an individual gps unit ( vxsport , lower hutt , new zealand , issue : 330a , firmware : 3.26.7.0 ) sampling at 4 hz and containing a triaxial acceloremter and a magnetometer in a total of 30 games . the gps unit ( mass : 76 g ; 48 mm x 20 mm x 87 mm ) was encased within a protective harness between the player s shoulder blades in the upper thoracic - spine region what ensured that players range of movement in the upper limbs and torso was not restricted . the system had been validated , deemed accurate and reliable during intermittent exercise ( buchheit et al . , 2014 ) . following each match gps data were downloaded using the same proprietary software ( vxsport view , new zealand ) . each file was trimmed so that only data recorded when the player was on the field was included for further analysis . the proprietary software provided instantaneous raw velocity data at 0.25 s intervals , which were then exported and placed into a customised microsoft excel spreadsheet ( microsoft , redmond , usa ) . the spreadsheet allowed analysis of distance covered ( m ) in the following categories : total distance ( td ) , high speed distance ( hsd : 17 kmh ) and sprint distance ( sd : 22 kmh ) . internal loading was measured as individualised training impulses ( itrimp ) ( manzi et al . , 2013 ) . pearson correlation coefficients were used to assess the relationships between ( 1 ) aerobic fitness and performance , and ( 2 ) distance : itrimp and fitness . qualitative interpretations of the correlation coefficients and the 95% confidence intervals ( ci ) as defined by hopkins ( 2004 ) ( 0.09 trivial , .10.29 small , .30.49 moderate , .50.69 large , .70.89 very large , .90.99 nearly perfect , 1 perfect ) are provided for all correlations . all statistical analyses were performed using spss for windows ( version 22 , spss inc . chicago , il , usa ) with statistical significance set at an accepted level of p<0.05 . the td , hsd and sd covered was 7604 510 m , 1623 149 m and 300 47 m , respectively . the internal tl ( itrimp ) of simulated match play was 534 74 au . the relationships between external load measures and fitness are shown in table 1 . specifically , td covered during the match simulation was not significantly correlated to aerobic fitness measures i.e. vlt ( r = -0.066 ; p = 0.747 ; 95% ci : 0.014 to 0.081 ; trivial ) , vo2max ( r = 0.152 ; p = 0.458 ; 95% ci : 0.114 to 0.261 ; small ) and vobla ( r = 0.014 ; p = 0.945 ; 95% ci : -0.014 to 0.084 ; trivial ) . hsd was not related to players vo2max ( r = 0.086 ; p = 0.677 ; 95% ci : 0.014 to 0.092 ; trivial ) and vobla ( r = 0.076 ; p = 0.712 ; 95% ci : 0.034 to 0.090 ; trivial ) . in contrast to previous observations , hsd was significantly related to vlt ( r = 0.485 ; p = 0.012 ; 95% ci : 0.314 to 0.619 ; moderate ) . finally , sd showed no significant correlation with vlt ( r = -0.189 ; p = 0.356 ; 95% ci : 0.014 to 0.091 ; trivial ) , vo2max ( r = 0.094 ; p = 0.647 ; 95% ci : 0.074 to 0.102 ; trivial ) and vobla ( r = 0.012 ; p = 0.955 ; 95% ci : 0.004 to 0.074 ; trivial ) . the integration of tl measures was shown to provide significant correlations with aerobic fitness measures . the relationship between tl integration ratios and aerobic fitness measures is shown in table 2 . specifically as shown in figure 1 , td : itrimp showed significant correlations with vo2max ( r = 0.524 ; p = 0.006 ; 95% ci : 0.224 to 0.754 ; large ) and vobla ( r = 0.559 ; p = 0.003 ; 95% ci : 0.314 to 0.774 ; large ) . similar trends were shown in figure 2 for hsd : itrimp and aerobic markers for fitness measures . p = 0.009 ; 95% ci : 0.214 to 0.861 ; large ) and vobla ( r = 0.407 ; p = 0.039 ; 95% ci : 0.204 to 0.641 ; moderate ) . finally , the integration of sd : itrimp shown in figure 3 was significantly related to vlt ( r = 0.611 ; p = 0.001 ; 95% ci : 0.414 to 0.841 ; large ) . table 1the correlation of the external tl with measures of aerobic fitnessexternal tlvo2max ( mlkgmin)vlt ( kmh)vobla ( kmh)total distance ( m)-0.158 - 0.0660.014high speed distance ( m)0.0860.4850.076sprint distance ( m)0.094 - 0.1890.012high speed distance ( m ; 17 kmh ) ; sprint distance ( m ; 22 kmh ) ; vlt = velocity at the lactate threshold ; vobla = velocity at onset of blood lactate accumulation**correlation is significant p= 0.012 table 2the correlation of external - internal tl ratios with measures of aerobic fitnessexternal : internal tl ratiovo2max ( mlkgmin)vlt ( kmh)vobla ( kmh)td : itrimp0.524 0.3300.599 hsd : itrimp0.3250.502 0.407 sprint : itrimp0.3580.611 0.170td : itrimp = total distance ( m):itrimp ( au ) ; hsd : itrimp = high speed distance ( m ) : itrimp ( au ) ; sprint : itrimp = sprint distance ( m):itrimp ( au ) ; vlt = velocity at the lactate threshold ; vobla = velocity at onset of blood lactate accumulation**correlation is significant p 0.05**correlation is significant p 0.05**correlation is significant p 0.05**correlation is significant p 0.05**correlation is significant p 0.05 figure 1a scatter plot showing the relationship between ( a ) td : itrimp and vobla ( kmh ) ( b ) td : itrimp and vo2max ( mlkgmin ) figure 2a scatter plot showing the relationship between ( a ) hsd : itrimp and vobla ( kmh ) ( b ) hsd : itrimp and vlt ( kmh ) figure 3a scatter plot showing the relationship between sd : itrimp and vlt ( kmh ) the correlation of the external tl with measures of aerobic fitness high speed distance ( m ; 17 kmh ) ; sprint distance ( m ; 22 kmh ) ; vlt = velocity at the lactate threshold ; vobla = velocity at onset of blood lactate accumulation correlation is significant p= 0.012 the correlation of external - internal tl ratios with measures of aerobic fitness td : itrimp = total distance ( m):itrimp ( au ) ; hsd : itrimp = high speed distance ( m ) : itrimp ( au ) ; sprint : itrimp = sprint distance ( m):itrimp ( au ) ; vlt = velocity at the lactate threshold ; vobla = velocity at onset of blood lactate accumulation correlation is significant p 0.05 correlation is significant p 0.05 correlation is significant p 0.05 correlation is significant p 0.05 correlation is significant p 0.05 a scatter plot showing the relationship between ( a ) td : itrimp and vobla ( kmh ) ( b ) td : itrimp and vo2max ( mlkgmin ) a scatter plot showing the relationship between ( a ) hsd : itrimp and vobla ( kmh ) ( b ) hsd : itrimp and vlt ( kmh ) a scatter plot showing the relationship between sd : itrimp and vlt ( kmh ) the current study investigated a novel approach of integrating external and internal workload variables as a ratio during hurling specific simulated match play . the results of the study show that measures of match running performance relate poorly to aerobic fitness measures . interestingly , there appears to be a relationship between markers of aerobic fitness and high speed running performance . the relationship has been previously reported in soccer populations ( akubat et al . , 2014 ) , but this is the first study to report this relationship in a hurling population . caution should be taken when utilising high speed running as a marker of performance , the training load and fatigue , given the match to match variability in this performance measure . gregson and colleagues ( 2010 ) reported that high speed running performance could vary by as much as 16% ( cv : 16.2 6.4% , 95% ci : 15.6 - 16.7% ) between competitive games 2015 ) observed large between - match ( within - player ) variation for high speed running distance ( 27.6% ; 90% confidence limits 6.9% [ forwards ] , 20.1% ; 4.1% [ backs ] ) , very high speed running distance ( 68% ; 19% , 34.1% ; 7.5% ) within rugby union . these findings question the reliability of these measures as accurate determinants of a player training load . the non - significant relationship between external load measures and measures of fitness seen in this study is in contrast to many studies that have analysed performance in other sports such as endurance running where the relationships between similar aerobic fitness variables in endurance events show very large to nearly perfect relationships ( mclaughlin et al . , 2010 ) . possible explanations of the non - significant relationships between the external measure of performance and fitness measures seen in this study can be explained by the simulated nature of match play . during competitive hurling match play greater effort may be required at a given time due to external stimuli such as standard of opposition , tactical formation or contextual factors ( paul et al . , 2015 ) . match simulations such as the hurling match play simulation protocol have been previously questioned as they allow players to pace themselves at levels that fail to replicate match play accurately . this pacing strategy can be related to the absence of opposition , tackling , tactical responsibility and game situation , all of which impact the locomotor performance and result in variability that may influence performance ( akubat et al . , 2014 ) . the impact of the above may explain the limited relationships between running performance and aerobic fitness variables observed during the current investigation . with the known limitations of analysing running performance , it may be more valuable to integrate both the external ( distance ) and internal ( itrimp ) load to provide a ratio that considers the internal load of exercise and the external output ( performance ) , thus providing a value representative of match day performance and fatigue ( akubat et al . , 2014 ) . previous studies have shown the value of the basic arbitrary itrimp unit as a marker of assessing adaptations in fitness throughout a pre - season period within professional soccer populations ( manzi et al . , 2013 ) . adding to this the integration method has been shown to have moderate to large correlations with aerobic fitness markers in a soccer population ( akubat et al . , 2014 ) . however , this study is the first to begin the process of integrating tl values in hurling . during the analysis td , hsd and sd athletes are provided with a valuable training and match day marker of the tl given banister s ( 1991 ) proposal that performance at a given time point is a result of fitness of an individual minus the accrued fatigue . the ratio provides the practitioner with a measure of the accrued fatigue at a given time point ( distance covered during the match or training session ) and the fitness measures ( itrimp ) . in addition , the temporal pattern of performance decrement has often been attributed to fatigue ( paul et al . , 2015 ) , therefore , this measure appears to have merits as a tl marker considering that it accounts for performance decrement and provides integration of this decrement with fitness measures . given the amateur nature of hurling players , frequent laboratory testing of fitness may be difficult . in addition , considering the known match to match variability in traditional external load measures such as td and hsd , the tl integration method may provide advancement in the assessment of the fitness if these measures of fitness predetermine a given performance and are reduced due to fatigue , it is reasonable to expect a performance decline . black and dobson ( 2011 ) showed that exercise induced fatigue reduced vo2peak and the ventilatory threshold showing a temporary reduction in measures of aerobic fitness . future research should be conducted during actual match play situations to assess if the integration ratio relationships hold true . moreover , there is a need for the assessment of these ratios as a marker of fatigue . by determining this , we can assess the validity of this ratio as a marker of the dose - response relationship as well as allow practitioners to become more proactive and manipulate the dose rather than reacting to the response . the current study presents additional findings to support the utilisation of integrated tl ratios in intermittent team sport environments . the findings suggest that the use of tl ratios provides data to practitioners that are correlated to aerobic fitness measures . in the current investigation , td was not significantly correlated to fitness measures i.e. vlt ( r = -0.066 ; p = 0.747 ; 95% ci : 0.014 to 0.081 ; trivial ) , vo2max ( r = 0.152 ; p = 0.458 ; 95% ci : 0.114 to 0.261 ; small ) and vobla ( r = 0.014 ; p = 0.945 ; 95% ci : -0.014 to 0.084 ; trivial ) . similar trends were seen with hsd with this external load measure not correlated to vo2max ( r = 0.086 ; p = 0.677 ; 95% ci : 0.014 to 0.092 ; trivial ) and vobla ( r = 0.076 ; p = 0.712 ; 95% ci : 0.034 to 0.090 ; trivial ) . interestingly , tl integration ratios were correlated to fitness measures more closely than external load measures . td : itrimp correlated with aerobic fitness measures i.e. vo2max ( r = 0.524 ; p = 0.006 ; 95% ci : 0.414 to 0.724 ; large ) and vobla ( r = 0.559 ; p = 0.003 ; 95% ci : 0.314 to 0.774 ; large ) . similar trends were shown for hsd : itrimp and aerobic markers for fitness measures i.e. vlt ( r = 0.502 ; p = 0.009 ; 95% ci : 0.214 to 0.861 ; large ) and vobla ( r = 0.407 ; p = 0.039 ; 95% ci : 0.204 to 0.641 ; moderate ) . the sd : itrimp showed significant correlations with vlt ( r = 0.611 ; p = 0.001 ; 95% ci : 0.414 to 0.841 ; large ) . overall , these measures support the use of integrated ratios as a method of monitoring training loads . integrated ratio measures were correlated to fitness measures , as such the use of these ratios presents advancement from the use of external load measures alone in the assessment of aerobic fitness of players .

structural genomic rearrangements are frequent findings in human cancers . therefore , papillary thyroid carcinomas ( ptcs ) were investigated for chromosomal aberrations and rearrangements of the ret proto - oncogene . for this purpose , primary cultures from 23 ptc have been established and metaphase preparations were analysed by spectral karyotyping ( sky ) . in addition , interphase cell preparations of the same cases were investigated by fluorescence in situ hybridisation ( fish ) for the presence of ret / ptc rearrangements using ret - specific dna probes . sky analysis of ptc revealed structural aberrations of chromosome 11 and several numerical aberrations with frequent loss of chromosomes 20 , 21 , and 22 . fish analysis for ret / ptc rearrangements showed prevalence of this rearrangement in 72% ( 16 out of 22 ) of cases . however , only subpopulations of tumour cells exhibited this rearrangement indicating genetic heterogeneity . the comparison of visual and automated scoring of fish signals revealed concordant results in 19 out of 22 cases ( 87% ) indicating reliable scoring results using the optimised scoring parameter for ret / ptc with the automated metafer4 system . it can be concluded from this study that genomic rearrangements are frequent in ptc and therefore important events in thyroid carcinogenesis . abstractthe current study aimed to assess the relationship between the hurling player s fitness profile and integrated training load ( tl ) metrics . twenty - five hurling players performed treadmill testing for vo2max , the speed at blood lactate concentrations of 2 mmoll-1 ( vlt ) and 4 mmoll-1 ( vobla ) and the heart rate - blood lactate profile for calculation of individual training impulse ( itrimp ) . the total distance ( td ; m ) , high speed distance ( hsd ; m ) and sprint distance ( sd ; m ) covered were measured using gps technology ( 4-hz , vx sport , lower hutt , new zealand ) which allowed for the measurement of the external tl . the external tl was divided by the internal tl to form integration ratios . pearson correlation analyses allowed for the assessment of the relationships between fitness measures and the ratios to performance during simulated match play . external measures of the tl alone showed limited correlations with fitness measures . integrated tl ratios showed significant relationships with fitness measures in players . td : itrimp was correlated with aerobic fitness measures vo2max ( r = 0.524 ; p = 0.006 ; 95% ci : 0.224 to 0.754 ; large ) and vobla ( r = 0.559 ; p = 0.003 ; 95% ci : 0.254 to 0.854 ; large ) . hsd : itrimp also correlated with aerobic markers for fitness vlt ( r = 0.502 ; p = 0.009 ; 95% ci : 0.204 to 0.801 ; large ) ; vobla ( r = 0.407 ; p = 0.039 ; 95% ci : 0.024 to 0.644 ; moderate ) . interestingly sd : itrimp also showed significant correlations with vlt ( r = 0.611 ; p = 0.001 ; 95% ci : 0.324 to 0.754 ; large ) . the current study showed that tl ratios can provide practitioners with a measure of fitness as external performance alone showed limited relationships with aerobic fitness measures .

medical experts consider skin - lightening and skin - bleaching practices as one of the most common forms of harmful body modification practices ( charles 2003 , p. 711 ) . melanie de souza argues that the desire for even - toned , blemish - free skin spans human populations . skin - lightening is just one of the multiple options for augmenting the skin s surface appearance , including but not limited to tanning , scarification , makeup , tattooing , face lifts , nose jobs , botox , lip extensions , and piercings . the desire to change skin colour from darker to lighter or from lighter to darker share an enhanced effect for the consumer , which has subjective and objective characteristics . this enhanced effect is directed to the way products are marketed that is to achieve a desirable skin colour or to improve an undesirable one . ironically , such an enhanced effect is overdetermined by inherent health risks , including skin cancers , steroid - induced acne , atrophy and telangiectasia . this practice can be traced back to colonialism , a period of institutionalised exploitation by colonists of indigenous populations . during colonisation privileged lighter skin with socioeconomic privilege . this paper traces historical events in south africa which may have aided or influenced the uptake and use of skin lightening creams . while globally , skin lighteners have been used for centuries , the earliest records of their use in south africa among black women suggest that it began in the 1950s alongside the coloured labour preference act of 1955 . this racially divisive legislation provided a distinct advantage to coloured individuals over black africans in relation to employment , accumulation of material wealth and property , and ease of movement , amongst other things . black women in the western cape province in particular , were more likely to find employment as domestic workers and cooks if they appeared to have a lighter complexion . in addition to the racial hierarchy of the apartheid state , colonial histories were dominated by subjective interpretations and morally - coded indictments of race and beauty . kenyasue smart argues that colonial vestiges cast european characteristics of whiteness with beauty and purity , while africans were characterised as demonic , ugly savages political , social and economic ideology premised on racial inferiority the legacy of apartheid in south africa coupled with westernised ideals of beauty underpins the desire to lighten as a means to overcome institutional forms of discrimination including colour stigma . former south african president thabo mbeki once referred to apartheid law ( 1948 - 1994 ) as poverty and the rule of race . apartheid was a system of governance premised on the oppression of dark - skinned populations by light populations . racial segregation occurred in almost every domain of public and private life ; wherein the minority white population received privileges and rights not available to the majority black population . simultaneously , the global media industry fuelled the notion that fair - skinned people were beautiful and thus , a standard was set for beauty . these factors together with trauma of colonization , slavery , discrimination , mistreatment , and colour rating in social class against dark skin continued to be further embedded in the psyche of those with more pigmented skin . in his book , black skin , white masks , franz fanon draws on his own experiences as a black person in a white dominated world . in his own struggles with stereotypes and prejudices fanon only wanted to be oneself with all the multiplicities , systems and contradictions of one s own ways of being , doing and knowing ( 1952 : pg . his work which can also be applied to the life of the american popular star michael jackson , describes the psychology of blackness and how notions of whiteness are internalized by colonizers as well as the manifestation thereof in the collective black psyche ( supplementary figure s1 ) . this manifests currently in the marketing of skin lightening products using terms such as bright , radiance , light and clear on the packaging because south african legislation prohibits the advertising of skin lightening products to bleach , lighten or whiten . all these factors continue to uphold white supremacy leading to a south african pigmentocratic society . paradoxically , the use of skin lightening creams increased after south africa achieved democracy in 1994 . as a result of liberation from apartheid - era legislation and racial classificatory schemes , new spaces for the expression of identity opened up . lightening in this way might be seen not in terms of black people aspiring to be white , but rather , free from the planters , where planters in this instance refers to the oppressors that rooted people to racial classificatory systems . global aesthetic regimes continue to place emphasis on , and preference for , lighter shades of brown . lighter shades of skin continue to inform beauty practices even in the context of black empowerment and afropolitan movements that continually seek to rupture colonial and western domination . consumer choice now fashions lightening as a form of human agency , where the surface of the skin can be manipulated to conform to , or resist global and local beauty standards . capitalism is thus implicated in the process of commodification identity politics . today , these socio - political influences are evident by a shared view , nahomie julien that a lighter shade of brown ( also referred to as yellow - bone ) is the most attractive skin colour . for example the magazine industry bears this out , with only one woman of colour appearing as one of the top 100 sexiest women in the global men s magazine , for him magazine ( fhm).the cosmetic industry unscrupulously advertises lightness to market products , which have the potential to endanger life . a conservative estimate proposes that 35% of south african women use skin lightening creams . in a more recent study , by ncoza dlova , which focused on the perception of possible benefits and risks associated with the use of skin lightening creams among africans and indians in durban , 32.2% of the population admitted to using skin lightening creams . the use of creams is prevalent across rural and urban demographics as well as across income and class stratification . studies located in asia , usa , u.k . , india , china and the caribbean indicate the prevalence of a thriving skin lightening cream industry . in three different african countries , the statistics demonstrate that 25% of women in bamako , mali a further 52% in dakar , senegal , and 77% in lagos , nigeria use skin lighteners . the underlying motivations to lighten skin in in south africa , as within an african context , drive our enquiry here . some people use skin lighteners to treat dermatological conditions such as hyperpigmentary melasma , age - induced darkening and acne . these treatments involve the use of hydroquinone ( hq)-containing products such as imiquimod but are under the strict guidelines of clinical use . some consumers use skin lightening products to lighten skin colour , enhance luminescence or radiance , even out skin tone , improve the texture of the skin , to satisfy peers , to satisfy ones partner and/or to attract partners and to enhance / increase employment opportunities . some studies have shown the impact of black celebrity endorsements for skin lightening products on consumer markets . for example , music celebrities like beyonce , rhianna and nicky minaj , give the impression that lighter skin is beautiful skin . even though none of these celebrities acknowledge the use of lightening products , there has been gradual lightening of their skin as can be observed in the media . while these celebrities can afford the most expensive product lines , average consumers tend to purchase creams at local suppliers , which do not comply with health regulations . some consumers make their own formulations from household products combined with skin lightening products , this in an attempt to enhance the whitening effect . this condition is characterised by localized , blue - black hyperpigmentation in the epidermis , dermis and subcutaneous layers of the skin . it is notifiable by progressive darkening in the area wherein the hydroquinone containing cream is applied . the efficacy of the majority of well - researched , marketed skin lighteners on sale at brand stores are effective when used correctly work well . the skin lighteners available in mainstream retailers are highly efficacious , but are marketed as skin toners or moisturisers . these formulations contain regulated amounts of hq and other ingredients such as retinoids and corticosteroids . according to current south african legislation , hydroquinone in skin lightening preparations it is crucial to note that the regulated formulations are sold with a product insert providing the necessary ingredient list and the correct usage , which should translate into the appropriate daily application . . lack of affordability may be one of the most significant drivers of the black market in south africa and africa where unregulated - and more affordable - products flood the market . due to the lack of product knowledge and absence of a product description , consumers rely on the reputation of the cream reinforced through positive word of mouth and consumer feedback . after the initial stages of application and resultant visible changes in complexion , consumers may apply more product than advised in a bid to make the product work faster and better . the consequence however results in irreversible damage to the skin , a condition known as exogenous ochronosis ( supplementary figure s1 ) . a more systemic , sustainable intervention is therefore needed at both the local and national governmental level before the colorism pandemic reaches exorbitant proportions . part of the solution would be an increased awareness through scientific and social engagement as well as harnessing the influential power of the media . the media s role in contributing to the positive outcome in the war against unregulated use of skin lighteners can not be overstated . the media plays an influential role in the purchase and use of skin lightening products . fairness can be portrayed as a sign of what is idealised and as a standard for beauty and competency . billboard advertising in africa for over 50 years portrayed white - skinned individuals as icons of beauty , as did the print and electronic media industries . today , in an even greater way , the media plays an influential role in how people live and how they perceive themselves . television , magazines , newspapers schoolbooks and the likes of social media platforms , facebook and twitter , ( supplementary figure s2 ) further emphasise the importance of image and beauty and how integral they are to achieving success . these platforms encourage the use of products as it presents visual stimuli where individuals with a light skin tone are portrayed as being more attractive and more likely to be successful in life . this ideology bolsters the perception that a fairer complexion is equated to material and social success ( supplementary figure s3 ) . advertisements promote the idea of fairness with slogans such as successful people , making your dreams come true , add sparkle to your life and lighter and lovelier . women claim that the way the television advertises skin lightening products , compels them to prefer lighter skin tones . commercials and advertisements for skin lightening products offer the promise of lighter , whiter skin . to tout the skin lightening products , advertisements are usually expensively produced , featuring ultra - light celebrities or super models , and presenting ( and mixing ) both traditional and westernized visual signifiers to evoke atmospheres of purity , cleanliness and sophistication ( leong , 2006 : pg . these forms of tele - marketing suggest black women are unhappy , ignored by men and they suffer from low self - esteem . ultimately , advertisements suggest , the lighter your skin , the more likely you are to find your dream occupation or even a suitable partner . when women are exposed to , or confronted with visual stimuli from the media , it creates a sense of anxiety and insecurity which results in them engaging in harmful forms of body beautification such as skin - lightening practices . well - known south african celebrities nomasonto mshoza maswanganyi ( supplementary figure s4 ) , a south african artist , bleached her skin because she was tired of being ugly ( drum , 17 november 2011 ) . other celebrities such as kelly khumalo , khanyi mbau and surisha naidoo have also been associated with lightening their skin . moving up towards upper africa the artist dencia has developed her own skin lightening product called whitenious where rumours also arise that she herself is using the product . in contrast to these celebrities encouraging skin lightening use , lupita nyongong , in her acceptance speech after winning an oscar embraced her blackness and spoke openly of her own insecurities over her dark skin and how she learned to love her skin . celebrities carry credibility and prestige , and their use of skin lighteners are perceived as being acceptable , even encouraged . consequently , consumers become obsessed with this practise because of the results obtained by its use and positive feelings associated with it . this can be attributed to the extent to which these products are marketed that is to improve appearance . this model tries to show that many variables , biological , psychological and social , interact to better understand and explain the reality of health and disease . adopting the biopsychosocial approach to or making a biopsychological review of the phenomenon of skin lightning means to underline or taking into consideration the interaction that exist between the three factors in all the process of skin bleaching : the motivation of doing it , the different practices and the effects of this practice , and even in the prevention and treatment ( support ) processes . this biological body is constructed on socio - cultural and psychological events of the individual . the skin is also a strong metaphor in the social sciences and embodies the realms of the aesthetic , political and economic , the social and cultural , and the genetic and phenotypic , as well as psychological worlds from an anthropological perspective , the skin has a physical reality as well as a social reality . the physical communicates shapes , sizes status which are then differentiated by culture . clothing on skin acts as protection even though some body parts are exposed . these shape , size and surface do have a social function which then applies to extreme forms of body mutilations such as tattooing , and piercings ( also known as cultural skins ) . these cultural skins also constitute them as being part of a group or some culture where they find a sense of belonging . helman also refers to people as social animals in the sense that they are organised into groups that regulate and perpetuate themselves . he argues that the persons experience as member of society that shapes his / her life of the world . in doing so it is believed that it is through one s culture that they organise and legitimizes their society . he describes culture as how humans organise themselves and the way they view the world which they inhabit . therefore in order to understand humans we need to study their society and their culture . terence turner s concept of social skin resonates in this case , wherein skin itself acts as an interface for political , cultural and social identity . the proliferation of skin - lightening creams emphasizes the importance of classification through skin - colour , stigma and preferences for lighter skin in an increasingly globalized world . he emphasizes that on a macro - social level , the conventionalized modifications of skin that comprise the social skin define , not individuals , but categories or classes of individuals thus claiming that the social skin becomes the boundary between social classes ( turner , 2012:503 ) . that means that the skin is an element of belonging , of classification , recognition , distinction and pride . skin bleaching is the use of cosmetics lightening products on the skin to look lighter . this practice sometime has negative effects as we said , and this effects side can have an impact on our body image and our self image the practice of skin - lightening may be deeply rooted in an individual s overall emotional and cognitive evaluation of his / her own worth ( self - esteem ) , their collections of belief about themselves and forces that influence thoughts , behaviour and personality . the desire to lighten one s skin is related to some aspects of self - hate ( extreme dislike or hatred against oneself ) and low self - esteem . even though colonialism and apartheid has the transgenerational psychological scars still persist and have been internalized by many . many women still regard lighter skin as beautiful and associated dark skin with negative connotations such as evil , disease , dirt ugliness etc . the hierarchy of women in terms of lightness of skin also known as pigmentocracy employs hegemonic ideals of beauty , influenced by the privileges of white supremacy established historically . many individual mimic the behaviour and attitudes of others with the hope of being like them . for example many women compare themselves with celebrities and turn to skin lighteners because they are dissatisfied with their appearance and are under the impression that they could be just as successful as the celebrities engaging in skin - lightening practices . in that sense skin - lightening practices can be perceived as an external ( social ) factor which dictates standards of beauty because having a light skin complexion is associated with elegance , beauty , attractiveness especially towards the opposite sex . lightness and darkness have moral connotations for example ; whiteness or lightness can be associated with youth , innocence , purity , virginity , spirituality and vulnerability , whereas darkness can refer to threat , aggression , danger , virility . individuals who are insecure , suffering from low self- esteem are more likely to engage in skin - lightening practices than those individuals who are more confident in their skin . skin lightening is a global phenomenon but has there been any attempt to reduce the practice of skin lightning ? despite the bans and existing health campaigns , the practice of skin lightening continues to grow . there is a need for greater government bans as well as control on the availability of skin lighteners , the marketing and sale thereof and well as the active ingredients found in these products . not only should government be involved , there should also be more awareness on the dangers associated with using skin lightening products . in addition social marketing in combating self - esteem issues has been proposed to reduce the risk that it may pose health risks , as they relate to skin alterations . social marketing is described as a tool that uses the concepts of commercial marketing to create positive social change . it is founded on the idea that media can shape popular perception and , thus promotes positive behaviour change by marketing ideas of products . therefore by promoting ideas around positive self - esteem , public health interventions can better address skin toning alterations . the history of skin- lightening practices were discussed by reviewing colonialism and apartheid as mediating factors in skin lightening use age . we also shed light on the reasons for skin lightening use and the dangers behind inappropriate application thereof . the media portray light skin desirable and beautiful and advertise skin lightening products as a means to solve problem skin . in their marketing strategies they use models , celebrities and high profile individuals to convey messages of lightness , beauty , wealth etc . the psychosocial effects on the consumer revealed that as a result of internalising the effects of colonialism and apartheid many individuals use skin lightening creams as a means to fit in the still much dominated white supremacy . lastly there is a great need for stricter government control and policy development in the marketing and distribution of products together with educational programmes to create awareness on the health risk posed by skin lightening products . interleukin 10 ( il-10 ) is a potent immunosuppressive cytokine made by regulatory t cells ( tregs ) and other cell types [ 13 ] . il-10 inhibits antigen - specific immune responses in part via suppression of activated macrophage and monocyte functions , which include cytokine synthesis and expression of class ii mhc and costimulatory molecules such as il-12 and cd80/cd86 . endogenous il-10 production is correlated with transplant acceptance in multiple animal models and human tissues [ 58 ] . il-10 has been evaluated as a treatment to improve the survival of engrafted islets , which has been accomplished by transfer of il-10-producing tregs , gene therapy [ 9 , 10 ] or direct administration of il-10 alone , or in conjunction with immunomodulatory drugs [ 1113 ] . it is noteworthy that systemic il-10 treatment has failed to support islet engraftment in mice in the setting of established autoimmunity and may induce immune suppression . these results suggest that an alternative approach that provides a sustained , local presence of il-10 at the graft site might be more effective at preventing islet rejection . we recently reported a role for the extracellular matrix ( ecm ) macromolecule hyaluronan ( ha ) in regulating il-10 production by t cells . ha is a simple , long - chain glycosaminoglycan polymer made up of repeating disaccharides of n - acetyl glucosamine and glucuronic acid . ha is an important structural component of many tissues , but also has important roles in inflammation and tissue repair [ 1518 ] . short ha oligomers ( < 30 kda ) generated through tissue catabolism are typically proinflammatory [ 1618 ] . conversely , plate - bound ha or chemically crosslinked ha is anti - inflammatory and promotes il-10 production by foxp3(+ ) natural tregs ( ntregs ) and conventional t cells in vitro . induction of il-10 in these systems was mediated by crosslinking of cd44 , the primary receptor for ha . we have proposed that plate - bound ha and ha hydrogels may function as biomimetics of ha - containing tissue matrices . however , the minimum size for ha - mediated cd44 crosslinking and il-10 production by t cells is unknown . additional support for a role for ha in il-10 production is provided by observations of ha - induced upregulation of il-10 by cultured synoviocytes and elevated il-10 levels in intestinal biopsies of mice given oral ha . however , ha alone does not appear to promote il-10 induction by t cells in vitro . indeed , our data suggest that concomitant antigenic stimulation through the t cell receptor ( tcr ) complex is required for efficient il-10 induction in the presence of ha . ha preparations are currently used as treatments for arthritis , atopic dermatitis , prevention of abdominal adhesions [ 25 , 26 ] and are under evaluation as an experimental treatment for burns and wounds [ 27 , 28 ] . in most of these preparations , crosslinking ( as well as plate - binding or sustained release from alginate ) may also limit the generation of pro - inflammatory ha fragments . building upon these findings , we have evaluated whether ha has utility in promoting il-10 production in vivo . here , we describe and evaluate a pair of technologies that both provide antigenic stimulation in the context of ha . first , we have asked whether cells implanted within a crosslinked ha hydrogel that incorporates a supplemental complex to induce polyclonal tcr stimulation could enhance production of il-10 in vivo . second , we have developed a bioengineered implant capable of delivering an antigenic signal along with sustained release of ha in fluid form . these technologies represent parallel strategies for delivering ha as a medium to promote il-10 production in vivo , with the ultimate objective of inducing durable immune tolerance to transplanted islets in individuals with autoimmune diabetes . c57bl/6 green fluorescent protein ( gfp)-foxp3 knock - in and ripmova / rag2 mice were the kind gifts of dr . a. rudensky ( memorial sloan - kettering cancer center , new york , ny , usa ) and dr . all mice were maintained in a specific pathogen - free , aaalac - accredited facility at bri , and all experiments were approved by the bri institutional animal care and use committee ( iacuc ) , protocol approval number 10116 . mouse lymphocyte populations were prepared as previously described . in brief , for the in vitro experiments , cd4(+ ) cells were isolated using macs kits ( miltenyi , inc . ) , and the gfp - foxp3( ) fraction was isolated from the cd4(+ ) population using a facs vantage cell - sorter ( bd biosciences ) . cd4(+)/gfp - foxp3( ) t cells were used to ensure that any il-10 production we measured would be from conventional t cells , rather than from activated gfp - foxp3(+ ) ntregs . cells were cultured in opti - mem ( invitrogen ) serum - free media supplemented with 100 g / ml penicillin and 100 u / ml streptomycin ( p / s ) . where specifically noted , cells were cultured in complete media consisting of dulbecco 's modified eagle 's medium ( dmem)-10 ( invitrogen ) supplemented with 10% fetal bovine serum ( fbs ) ( hyclone ) , p / s , 50 m -mercaptoethanol , 2 mm glutamine , and 1 mm na pyruvate ( invitrogen ) . cell culture plates ( 96-well ) were coated with 0.5 g / ml of anti - cd3 antibody ( 145 - 2c11 , bd biosciences ) , washed , and then subsequently coated with either 0.2 mg / ml bovine serum albumin ( bsa)- conjugated ha ( 1.5 10 da ) ha ( genzyme ) or 10% bsa . cd4(+)/gfp - foxp3( ) t cells ( 2 10 per well ) were cultured for 96 hours on these plates , followed by collection of the culture supernatants for analysis . measurement of cytokines in the cell culture supernatants was performed using enzyme - linked immunosorbent assays ( elisas ) or cytometric bead assays ( bd biosciences ) . hydrogels were made from thiolated constituents ( ha , heparin sulfate [ hs ] , and collagen ) crosslinked with polyethylene glycol s - s diacrylate ( pegssda ) . these reagents are available as a kit ( extracel - hp , glycosan / biotime ) and were used per the manufacturer 's instructions . of note , our understanding from communications with the manufacturer is that ha of > 1 10 da is used in the kits . prior to addition of the crosslinker , the mixture was supplemented with 10 g / ml of streptavidin ( sigma aldrich ) , 10 g / ml each of biotinylated anti - cd3 and anti - cd28 antibodies ( 145 - 2c11 , 37.51 , bd biosciences ) , and 20 iu / ml of il-2 . hydrogels of this formulation are referred to here as supplemented ha hydrogels . for in vitro cell culture experiments , 2 10 cd4(+)/gfp - foxp3( ) t cells were layered on top of 25 l volumes of the hydrogel . after 96 hours of culture , cells and culture supernates were collected for analysis . to control the collagen constituent of the ha hydrogels , a set of hydrogels lacking ha / hs was made by replacing the thiolated ha / hs with an equivalent volume of thiolated collagen . 3 10 cd4(+)/gfp - foxp3( ) t cells were dispersed in supplemented ha hydrogels of 300 l volume prior to crosslinking with pegssda , which was initiated 30 min prior to intraperitoneal injection into mice . for these studies in vivo , the supplemented ha hydrogels incorporated 360 iu / ml of il-2 . four days after injection of the supplemented ha hydrogels , the mice were sacrificed and lymphoid tissues were harvested . residual hydrogel material in the peritoneal cavity was also removed and dissolved by mild reduction of the pegssda ( per the manufacturer 's instructions ) in order to retrieve cells for analysis . intracellular staining of these cells for il-10 and subsequent flow cytometry assays utilized antibodies and equipment as previously described . briefly , c57bl/6 mice of 1224 weeks age were anesthetized with 2,2,2-tribromoethanol in phosphate - buffered saline ( pbs ) . the descending aorta of each anesthetized mouse was transected , the bile duct clamped at its distal ( intestinal ) end , and a 30-gauge needle was used to inflate each pancreas through the common bile duct with 4 ml of 4c islet medium comprised of rpmi 1640 containing 1.0 g nahco3 , 10% fbs ( atlanta biologicals , cat . number s12450h ) , 1 mm na - pyruvate , and p / s . the islet medium was supplemented with 0.8 mg / ml of collagenase p ( roche , cat . number 11 - 249 - 002 - 001 ) and filtered at 0.22 m prior to injection . subsequently , 2 - 3 excised pancreata were placed in separate 50 ml conical centrifuge tubes and incubated in 5 ml of islet medium for 13 min at 37c . the medium was then decanted , fresh 4c islet medium was added , and the tubes were shaken vigorously to disrupt the pancreata . the tissue suspensions were filtered through a 30-mesh metal screen to remove large debris , the filtrates were pelleted by centrifugation , and the pellets resuspended in 4c islet medium . the resuspended material was centrifuged through histopaque 1077 to isolate the islets , which were washed , resuspended in islet medium , and placed in a tissue culture ( tc ) incubator . after all pancreata were processed , the isolated islets were hand picked , cultured overnight , and picked again the next day before being placed in bioengineered implants . biopsy punches ( sklar instruments ) were used to cut 10 mm diameter disks from 2 mm thick sheets of pva sponge ( type cf90 , 500 m average pore size with no surfactant treatment a generous gift from merocel / medtronic , inc . ) . subsequently , each disk was through - punched with a single central hole of 2 mm diameter and six peripheral holes of 1.5 mm diameter , using correspondingly sized biopsy punches ( acuderm , inc . ) . the punched disks were washed 5 10 min on a rocker in 50 ml centrifuge tubes filled with 40 ml of sterile distilled water , then air - dried on whatman filter paper , transferred to 60 mm dishes , sterilized by gamma irradiation , and stored until needed for bi assembly . one volume of a stock solution of rat tail native type i collagen in dilute ( 0.02 n ) acetic acid ( bd biosciences ) was combined with 1/9 volume of 10-strength nahco3-saturated medium 199 ( invitrogen ) and sufficient dmem and normal mouse serum ( nms ) to yield a working solution containing 2.5 mg / ml collagen and 10% nms . the working solution was prepared just prior to assembly of the bis and kept on ice until needed . number a0682 ) , filtered at 0.45 m , was used for preparation of spheres for sustained release of vascular endothelial growth factor ( vegf ) and ha . briefly , a mixture of 2% alginate and 5 ng/l vegf was formed into 10 l ( 2.2 mm diameter ) spheres using a gravity - drop method , crosslinked into a hydrogel for 15 min in 0.1 m cacl2 , washed 2 2 min in 0.15 m nacl/25 mm hepes/2 mm cacl2 , ph 7.2 ( saline / hepes / ca ) , transferred to serum - free dmem / p / s , and kept in a tissue culture incubator until needed for bi assembly . fabrication of ha spheres was similar to that of the vegf constructs , with replacement of the vegf with 50 g of 120 kda ha ( genzyme ) . ha of this size ( approximately 317 disaccharide units ) was chosen to facilitate a complete delivery of ha from the spheres within a 2 week experimental time period . dry pva sponge scaffolds were allowed to swell for 5 min in sterile dmem / p / s . subsequently , a single , freshly prepared alginate sphere containing vegf and five spheres containing ha were gently pressed into the 6 peripheral holes of each expanded scaffold . the scaffolds were then blotted on sterile whatman filter paper , transferred to 60 mm plastic cell culture dishes lined with uv - sterilized parafilm m , and flooded with 60 l of type i collagen working solution containing suspended islets . the pva sponges absorbed the collagen solution , with the majority of the islets entering the 2 mm diameter central hole of the scaffold . subsequently , the dishes were covered with dish tops ( lined with moist filter paper ) and placed in a tissue culture incubator for 30 min to polymerize the collagen into a hydrogel . the completed bis were placed in dmem/10% nms / p / s in 24-well cell culture plates and kept in a tissue culture incubator prior to implantation in mice . to measure the kinetics of release of ha from alginate hydrogels in vitro , spheres containing 2% alginate and 2.5 g of fluorescein isothiocyanate ( fitc)-conjugated 120 kda ha or 1.5 10 da ha were prepared as described previously . the spheres were placed in 96-well cell culture plates ( one sphere per well ) that had each of the wells filled with 200 l of dulbecco 's ca / mg pbs ( dpbs ca / mg , invitrogen ) . the plates were placed in a tissue culture incubator , and 100 l volumes of the media were removed from each well at specific time points ( up to 14 days ) and analyzed by fluorescence spectrophotometry to quantify released fitc - ha , using a standard curve of fluorescence versus known concentration of fitc - ha . following removal of the medium at each time point , the residual medium in each well was discarded , and each well was refilled with 200 l of fresh medium . to determine the percentage of ha retained in alginate spheres during their fabrication , freshly prepared spheres containing 2.5 g of fitc - ha were dissolved in pbs/100 mm ethylenediaminetetraacetic acid ( edta ) , and the resultant solution was analyzed by fluorescence spectrophotometry to quantify total fitc - ha per sphere . bis were implanted into mesenteric pockets of ripmova / rag2 mice ( one bi per mouse ) using previously described protocols , followed by injection of the mice 24 hours later with 1 10 purified ova - specific cd4(+ ) do11.10 t cells we previously demonstrated that plate - bound ha together with an antigenic signal promotes il-10 production by cd4(+)/gfp - foxp3( ) t cells . this led us to ask whether we could develop this finding into a tool for use in promoting il-10 production in vivo . to this end , we modified a ha - based hydrogel to deliver a polyclonal antigenic stimulus through addition of streptavidin , biotinylated anti - cd3/cd28 antibodies , and il-2 . we have previously shown that a similar form of supplemented ha hydrogel is an efficient way to elicit il-10 production from t cells in vitro . we found that cd4(+)/gfp - foxp3( ) t cells exposed to the supplemented ha hydrogels produced il-10 at significantly higher levels than did corresponding t cells activated with anti - cd3/cd28 antibodies and il-2 on cell culture plates ( figure 1(b ) ) . this was the case whether the cells were cultured on top of the gels ( as shown ) or embedded within the gels ( data not shown ) . omission of either streptavidin or anti - cd3 antibody from the gel mixture likewise abrogated il-10 production ( data not shown ) , indicating that cd3 was required for the stimulus and suggesting that streptavidin was necessary to retain cd3 in the hydrogel lattice . streptavidin , biotinylated anti - cd3/cd28 antibodies , and il-2 incorporated into a hydrogel lacking ha ( supplemented collagen hydrogel ) did not significantly increase il-10 production over plate - bound activation by these agents ( figure 1(b ) ) , which demonstrated the potentiating influence of ha on il-10 production . the unique capability of ha to stimulate il-10 production by t cells is underscored by the observation that hydrogels made from other types of ecm , including basement membrane components ( matrigel ) and fibrin , are not stimulatory in vitro . to evaluate whether supplemented ha hydrogels could be used to induce il-10 production in vivo , the gels were populated with 3 10 cd4(+)/gfp - foxp3( ) t cells from cd45.2 mice and injected into the peritoneal cavities of cd45.1 mice . by use of the cd45.1 and cd45.2 allelic markers four days after implantation , spleens and lymph nodes were harvested , processed , the released cells stained for intracellular il-10 and cd markers , and gating performed to distinguish donor t cells from host t cells ( figure 2(b ) ) . as controls , analogous supplemented collagen hydrogels lacking ha were populated with cells and injected into a designated set of mice . after 4 days , a substantial volume of residual ha hydrogel was found within the peritoneal cavities of the treated mice ; however , the control collagen hydrogels had dissolved . in separate experiments , we found that after 7 days no implanted ha hydrogels were identifiable , indicating that extensive catabolism of the hydrogels takes place in vivo . the cells within the ha hydrogel residue 4 days after implantation were primarily cd45.2(+ ) and expressed il-10 at a high level relative to host t cells from the spleen ( figure 2(c ) ) . these cells remained foxp3( ) ( data not shown ) , consistent with our previous report that ha does not induce foxp3 expression . cell isolates from the spleens and lymph nodes of the transplanted mice contained cd45.2(+ ) donor t cells ( figure 2(d ) ) , which indicated that the t cells embedded in the hydrogels had migrated into lymphoid tissues . donor t cells that migrated from the supplemented ha hydrogels expressed higher levels of il-10 than the corresponding donor t cells that migrated from the control collagen hydrogels . host t cells from these two groups of mice did not express il-10 above levels of the nonspecific antibody controls ( figure 2(d ) ) . these data indicate that ha hydrogels providing endogenous tcr stimuli can be used as platforms to induce il-10 production in vivo . while supplemented ha hydrogels are a novel system for inducing implantable t cell populations that produce il-10 , we sought to devise an implantable platform that would elicit il-10 production from endogenous t cells in an antigen - specific manner . to this end , we adapted a novel bioengineered implant ( bi ) we had developed from an earlier study to combine the antigenic stimulus with sustained release of ha within the same construct . we recently reported on the development of the bi as a model system to explore improved approaches for islet transplantation . the bi , sized for mesenteric or subcutaneous implantation in mice , consists of a disk - shaped pva sponge infused with a type i collagen hydrogel that contains dispersed donor islets . to promote islet vascularization previously , we used syngeneic mice to demonstrate that bis containing 450500 islets and 20 ng of vegf could reverse streptozotocin ( stz)-induced diabetes in 100% of recipients . notably , none of these mice required exogenous insulin therapy once the bis began to fully regulate levels of blood glucose . moreover , the transplanted mice responded to glucose challenge in a near - normal manner . induction of pro - tolerogenic cytokines , such as il-10 , is an appealing strategy to help facilitate transplantation of islets . here , we have adapted our bi device to evaluate the capacity of ha in fluid form ( i.e. , ha not crosslinked to form a hydrogel ) to elicit il-10 production in an autoimmune setting . to test this model , we loaded the bi with islets expressing the ova antigen , transferred in ova - specific t cells , immunized the recipient mice with ova , and asked whether these cells expressed il-10 in an ova or tcr - specific manner . we first evaluated the kinetics of release of ha from 2 mm diameter , 2% alginate spheres under physiological conditions in vitro ( figure 3 ) . we found that release of 1.5 10 da ha was linear , but relatively slow in contrast , the release of 120 kda ha was much more rapid essentially 100% was released within 14 days , which was a useful time frame in which to analyze post - transplantation immune responses . we found that over 60% ( 63.7% 6.1% ) of the 120 kda ha loaded into each sphere was retained by the alginate after crosslinking with calcium . the bis we designed for our experiments in vivo ( figure 4 ) incorporated a single alginate sphere containing 20 ng of vegf and 5 spheres that contained a total of 160 g of 120 kda ha ( the total is derived from a value of 32 g of ha per sphere , based on 64% retention of the 50 g of ha present in each sphere prior to calcium crosslinking ) . a set of control bis incorporated one vegf sphere and 5 spheres loaded with saline in place of the ha . the central hole of the bi was infused with a type i collagen hydrogel containing islets from ripmova mice , which express chicken ovalbumin driven by the rat insulin promoter ( rip ) . bis of this design were implanted into mesenteric pockets of ripmova / rag2 mice ( one bi per mouse ) , followed by injection of the mice 24 hours later with 1 10 purified ova - specific cd4(+ ) do11.10 t cells . forty - eight hours after implantation , each mouse was immunized with 100 g of ova peptide ( aa 323339 ) . on day 14 after implantation , splenocytes were isolated from the mice and assayed for il-10 production in vitro after 96 hours of stimulation with either anti - cd3/anti - cd28 antibodies or antigen - specific ova peptide ( figure 5(a ) ) . in this assay , the splenocytes from the mice treated with ha produced more il-10 than the splenocytes from the control mice that were not exposed to ha . this differential response was observed when the splenocytes were given either a nonspecific stimulus with cd3/cd28 ( figure 5(b ) ) or a specific stimulus with ova peptide ( figure 5(c ) ) . unfortunately , these data do not allow us to discern which cells are the source of il-10 in this assay and specifically whether the cells in question are foxp3(+ ) tregs or foxp3( ) conventional t cells . we demonstrate , using two separate model systems , that delivery of ha together with antigenic signals promotes the production of il-10 in vivo . our data suggest a potential clinical application for ha - mediated induction of il-10-producing t cells using injectable hydrogels . ha hydrogel platforms are in development for a variety of applications , including drug delivery and wound dressings , and are noted for their biocompatibility [ 31 , 32 ] . in the present study , we have shown that augmentation of ha hydrogels with a complex of biomolecules that provide tcr stimulation in addition to the ha signal can deliver the requisite cues for il-10 induction , both in vitro and in vivo . in treatments of diabetic patients that involve transplantation of islets , controlling rejection levels must be low enough to permit a reasonable degree of protective immunity against pathogenic organisms , but high enough to effectively suppress allo- and autoimmune activity . in the case of simultaneous pancreas - kidney ( spk ) transplants , some current immunosuppression regimens are inadequate to control autoimmunity [ 33 , 34 ] . moreover , no matter what the dose , systemic immunosuppression can be accompanied by a variety of undesirable side - effects on tissue and organ systems that are not directly associated with the transplant . in light of the problems associated with systemic treatments , an alternative approach would be to confine the delivery of immunotherapy to the implant itself . in this way , immunomodulatory compounds could be delivered at relatively high concentrations , but within the limited volume of the implant , thereby minimizing side - effects on tissues and organs outside the zone of delivery . to this end , the bi described here includes a mechanically - supportive scaffold and ecm hydrogel that concentrates the islets in a small volume , and a sustained - release component for local delivery of immunomodulatory compounds . in the present study , we have adapted our bi to release ha in fluid form . rather than using ha of 11.5 10 da that is typically incorporated into ha hydrogels , we used ha with a 10-fold lower mw ( 120 kda ) to provide release kinetics that were optimal for the 14-day duration of our experiments in vivo . of note , we observed that this shorter ha could induce an il-10 response from host mice . to our knowledge , this observation is the first demonstration of il-10 production by ha of this weight class . it is possible that the 120 kda ha is crosslinked into higher mw forms after its release into tissue from alginate , which could be accomplished by ha - binding molecules such as inter - alpha - trypsin inhibitor ( ii ) and/or tumor necrosis factor - stimulated gene-6 protein ( tsg-6 ) which are present at sites of inflammation and which are known to crosslink ha into macromolecular assemblies [ 3537 ] . such crosslinking could result not only in a functional increase in the mw of ha , but also promote the retention of ha in the fibrovascular tissue that invades the bi and in the tissues immediately surrounding the implant . our future studies will continue to use bis as platforms to evaluate the effectiveness of ha and other specific ecm and cytokine environments on islet survival and reversal of diabetes in the setting of autoimmunity .

skin - lightening is an aesthetic practice of global concern . by adopting a biopsycho - social approach , we consider the interplay between the biological , psychological and social factors that underpin the circulation and consumption of skin lighteners in south africa . this paper reflects on biological aspects of skin lightening , interpersonal relationships , individual beliefs and expectations about the maintenance of health and well being that informs cosmetic practices . the paper seeks to examine claims made by historians ( thomas ) and political philosophers and activists ( biko ) that colonialism and apartheid in south africa historically reinforced the use of skin lightening products in the country . the paper also investigates the role of media in staking out the boundaries of beauty . we argue that men and women practice skin - lightening not only as a complex result of the internalization of global standards of beauty , but meshed with a national politics of race and colorism . banning skin lightening products without understanding the biological effects but also the social forces that underlie their increased popularity will prove futile . moreover , we must consider the immeasurable pleasures associated with lightening , and the feelings with achieving visibility in south africa , a country that continues to wrestle with blackness . local induction of pro - tolerogenic cytokines , such as il-10 , is an appealing strategy to help facilitate transplantation of islets and other tissues . here , we describe a pair of implantable devices that capitalize on our recent finding that hyaluronan ( ha ) promotes il-10 production by activated t cells . the first device is an injectable hydrogel made of crosslinked ha and heparan sulfate loaded with anti - cd3/anti - cd28 antibodies and il-2 . t cells embedded within this hydrogel prior to polymerization go on to produce il-10 in vivo . the second device is a bioengineered implant consisting of a polyvinyl alcohol sponge scaffold , supportive collagen hydrogel , and alginate spheres mediating sustained release of ha in fluid form . pancreatic islets that expressed ovalbumin ( ova ) antigen were implanted within this device for 14 days into immunodeficient mice that received ova - specific do.11.10 t cells and a subsequent immunization with ova peptide . splenocytes harvested from these mice produced il-10 upon re - challenge with ova or anti - cd3 antibodies . both of these devices represent model systems that will be used , in future studies , to further evaluate il-10 induction by ha , with the objective of improving the survival and function of transplanted islets in the setting of autoimmune ( type 1 ) diabetes .

long term use of chronic oral anticoagulants can be complicated by anticoagulation associated intracranial hemorrhage ( aaich ) , the most devastating complication of anticoagulant therapy . among patients who received anticoagulant for management of thromboembolic problems , intracranial hemorrhages led to approximately 90% of deaths and major disability in survivors5 ) . rapid reversal is essential for prevention of enlargement of the aaich , which could result in brain herniation . on the other hand , reversal and discontinuance of anticoagulant can result in development of several kinds of side effects , such as thromboembolic complication . the risk and incidence of thromboembolic complication after cessation of anticoagulation in korean society have not been accurately reported ; in addition , there is no consensus regarding the proper period for withholding anticoagulant in the case of these patients . based on results of other studies , discontinuation for seven to 10 days appears to be safe2 ) . however , this has limitation as a proper guideline because these data were reported from small western retrospective series3,6,11 ) . in this study , we discussed reasonable access for management of patients with aaich who are at a high thromboembolic risk in the view of using an anticoagulant . from january 2008 to december 2012 , 47 patients with warfarin associated intracranial hemorrhage were admitted to a single neurological institution . eleven patients ( 23.4% ) died due to an initial massive hematoma and were excluded from this study . of the remaining 36 patients , 19 patients were not restarted with anticoagulant due to high risk of rebleeding and 17 patients were restarted with anticoagulant at a different time depending on each patient 's condition . restarting of drug was decided after consultation with the doctor who treated the patients due to the underlying disease . however , they did not give a clear answer regarding when the drug should be restarted after hemorrhage . if the rebleeding risk was greater than the thromboembolic risk , anticoagulants were not restarted . for these reasons , underlying disease to get oral anticoagulant , presenting hemorrhage type , thromboembolic risk factors , event day international normalized ratio ( inr ) , prothrombin time ( pt ) , and thromboembolic complication after cessation of anticoagulation were investigated . data analysis was performed retrospectively using the statistical package for the social sciences ( spss ) version 20.0 ( spss inc . , descriptive statistics were performed for determination of the influence of clinical factors on development of thromboembolic events in the non - restarted anticoagulant group . the kaplan - meier method was used to assess the incidence and probability of thromboembolic events in patients with passage of time . we need to identify the exact degree of thromboembolic risk in the patients ' group in order to obtain objective validity when comparing the results with those of other authors . in this study the cha2ds2-vasc [ congestive heart failure / left ventricular dysfunction , hypertension , age 75 ( double ) , diabetes , stroke ( double)-vascular disease , age 65 - 74 , and sex category ( female ) ] has been used in multiple cohorts and is better at identifying patients with true risk . the score is inclusive of the most common risk factors for stroke in everyday clinical practice4,8 ) . from january 2008 to december 2012 , 47 patients with warfarin associated intracranial hemorrhage were admitted to a single neurological institution . eleven patients ( 23.4% ) died due to an initial massive hematoma and were excluded from this study . of the remaining 36 patients , 19 patients were not restarted with anticoagulant due to high risk of rebleeding and 17 patients were restarted with anticoagulant at a different time depending on each patient 's condition . restarting of drug was decided after consultation with the doctor who treated the patients due to the underlying disease . however , they did not give a clear answer regarding when the drug should be restarted after hemorrhage . if the rebleeding risk was greater than the thromboembolic risk , anticoagulants were not restarted . for these reasons , underlying disease to get oral anticoagulant , presenting hemorrhage type , thromboembolic risk factors , event day international normalized ratio ( inr ) , prothrombin time ( pt ) , and thromboembolic complication after cessation of anticoagulation were investigated . data analysis was performed retrospectively using the statistical package for the social sciences ( spss ) version 20.0 ( spss inc . , descriptive statistics were performed for determination of the influence of clinical factors on development of thromboembolic events in the non - restarted anticoagulant group . the kaplan - meier method was used to assess the incidence and probability of thromboembolic events in patients with passage of time . we need to identify the exact degree of thromboembolic risk in the patients ' group in order to obtain objective validity when comparing the results with those of other authors . in this study the cha2ds2-vasc [ congestive heart failure / left ventricular dysfunction , hypertension , age 75 ( double ) , diabetes , stroke ( double)-vascular disease , age 65 - 74 , and sex category ( female ) ] has been used in multiple cohorts and is better at identifying patients with true risk . the score is inclusive of the most common risk factors for stroke in everyday clinical practice4,8 ) . the demographic and clinical characteristics of 19 patients in whom anticoagulants were not started are shown in table 1 and 2 . the most common type of aaich was spontaneous hemorrhage , which presented in eight cases ( 42.1% ) . acute subdural hematoma was the next main type of aaich in five cases ( 21.1% ) . the mean discontinuance time of the anticoagulant was 475.79682.49 days ( range , 8 - 1813 days ) . there were eight cases ( 42.1% ) with thromboembolic complications , three of which ( 37.5% ) resulted in death . two deaths were caused by sudden cardiac arrest , which was suspected with acute myocardial infarction , and another patient died of pulmonary thromboembolism . among the survivors , deep venous thrombosis followed by intravenous filter insertion occurred in two cases and another two patients presented with angina pectoris . and non - fetal pulmonary embolism occurred in one case . mean time to occurrence of thromboembolic complication was 21.3814.89 days ( range , 8 - 56 days ) . the probabilities of having thromboembolic complications at 7 , 14 , and 30 days following cessation of warfarin therapy were 0.0% ( 95% confidence interval , 0.00 - 20.92% ) , 10.53% ( 95% confidence interval , 1.84 - 34.54% ) , and 38.49% ( 95% confidence interval , 18.42 - 62.85% ) , respectively ( fig . the inr showed a gradual decrease to the normal range , and then thromboembolic complications started to occur after a few days . from the result , we found that all thromboembolic complications and death occurred after seven days in all cases , and most of these thromboembolic complications tended to be concentrated between one and two weeks . this study provided the longest follow up data on discontinuance of anticoagulant in patients presenting with aaich who have been at high thromboembolic risk estimated by cha2ds2-vasc score in korean society . first , discontinuance of the anticoagulant is common in aaich patients in the actual clinical field . proper restart of warfarin treatment after aaich was not prescribed for almost half of patients ( 47.2% , 17/36 ) . aaich induced mortality is very high and survivors from initial neurological damage are faced with the decision of whether or not to restart anticoagulan3,5 ) . however , proper use of anticoagulant in patients with aaich has not yet been established , and guidelines could not be obtained from a limited number of previous retrospective series3,6,11 ) . according to american heart association recommendations , in patients with a very high risk of thromboembolism in whom restarting warfarin is considered , warfarin therapy may be restarted at seven to 10 days since onset of the original hemorrhage2 ) . in this study , the consensus of the resumption of anticoagulant in aaich patients is not clear in our institution . decisions regarding to the starting time of anticoagulant were differed significantly among physicians due to the fact . and available data for determination of the optimal timing and dosage for restarting anticoagulation following an episode of aaich are inadequate . second , in this study , short duration ( within 7 days ) discontinuance of anticoagulants was relatively safe , similar to the result of a previous study series . kawamata et al.7 ) found no cases of ischemic stroke when warfarin treatment was discontinued for three days in 27 patients with aaich . tinker et al.10 ) retrospectively reviewed the risk of discontinuing warfarin therapy in 159 patients with mechanical heart valves undergoing elective surgery ; none of the patients had in - hospital thromboembolic complications . ananthasubramaniam et al.1 ) also reported that no thromboembolic complications occurred in 24 patients with prosthetic heart valves after withholding anticoagulant for an average of 15 days . like these previous studies , in this study , there was also no occurrence of thromboembolic complications within seven days since aaich . although a temporary withdrawal of anticoagulants within seven days may be safe , prolonged cessation could lead to catastrophic results . we found that thromboembolic complications begin to occur after seven days and the incidence shows a rapid increase around 14 days later . however , additional events occurred after two weeks . overall occurrence of thromboembolic complications was 42.1% ( 8/19 ) , higher than that reported in previous series1 ) . first , the time for withdrawal of anticoagulant was long , compared to the previous reports . in previous studies1 ) , anticoagulant was restarted within at least two weeks after a hemorrhagic event . the inr in patients with thromboembolic complication showed a gradual reduction and it fell below the normal range on ictus days . because the thromboembolic risk was accumulated after cessation of anticoagulants , second , the patients belonged to a very high risk group for occurrence of thromboembolic events . the cha2ds2-vasc score has been used in multiple cohorts for measurement of severity and for better identification of patients with true risk4 ) . the score includes the most common risk factors related to stroke in everyday clinical practice . among patients with cha2ds2-vasc score=2 , the incidence of thromboembolic complication in one year is 2.69% and the rate increased by 3.20% at a score of 3 points . for prevention of thromboembolism , anticoagulants are recommended for patients with a score of more than 2 points9 ) . in this study , the cha2ds2-vasc score was more than 2 points for all patients ( 2 points in three cases , 3 points in five , and 4 and 5 points in one case each ) , indicating that the patient has a high underlying thromboembolic risk . otherwise , previous reports did not objectively demonstrate the patients ' underlying thromboembolic risk , causing difficulty in comparison and analysis of results between studies . the limitation of this study is the retrospective design with a very small number of cases . however , the incidence of patients who did not restart anticoagulation after aaich was very low . over the past five years discontinuance of anticoagulant within seven days presented a relatively low incidence of thromboembolic complications in patients with aaich at a high thromboembolic risk ( cha2ds2-vasc score > 2 ) , and withdrawal of more than seven days may increase the risk of devastating results . we think that short term discontinuance of anticoagulant limited within seven days appears to be safe in korean people . unfortunately , this study was conducted retrospectively in a small number of cases of aaich and has several limitations to understanding . for a more reasonable and acceptable guideline on the use of anticoagulant in patients with aaich , conduct of further studies will be required . chronic fluoride poisoning is a global health problem that occurs endemically in areas where the fluoride content in water is above the optimal level . world health organization 's ( who ) international standards of 1958 and 1963 referred to fluoride for drinking water , claiming that consumption of water with fluoride concentrations above 1.01.5 mg / l can result in pathological changes in teeth causing dental fluorosis ( df ) , which is characterized by light yellow to brown black horizontal lines on the teeth surface and chipped off edges . high concentrations of fluoride can also produce long - term bone damage in children and adults such as skeletal fluorosis . traditionally , dental fluorosis has been connected with a higher intake of fluoride coming from drinking water , which may contain high fluoride concentrations , especially in groundwater of areas of volcanic rocks . these high - risk areas are mostly located in arid and semi - arid regions that are characterized by a rapid rate of chemical weathering of geological materials . according to the who , permissible fluoride concentration limit in drinking water is 1.0 mg / l , and mexican normativity stipulates a limit of 1.5 mg / l . nonetheless , in mexico , there are some areas that have high amounts of fluoride in water , mainly in the states of the north and center of the country , most notably in chihuahua , durango , aguascalientes , and guanajuato . fluoride ingested remains for a long time in the human body , however , approximately 80% of fluoride entering the body is excreted mainly through urine ; the rest of it is absorbed into body tissues from where it is released very slowly . excreted fluoride can be monitored by biomarkers of fluoride , which are values that serve to identify deficient or excessive consumption and bioavailability of fluoride in the body . who defines different fluoride biomarkers ; current ( urine , plasma , and saliva ) , recent ( nails and hair ) , and historical biomarkers ( bones and teeth ) . urine fluoride concentration among the biomarkers of fluoride exposure is generally accepted as the best indicator of fluoride exposure because it can be recollected noninvasively and systematically reflects the burden of fluoride exposure from drinking water . hence , special attention has been given to it as a biomarker , and is used as an indirect indicator of fluoride intake . official mexican norms ( nom-013-ssa2 - 1994 ) stipulates , periodically monitoring urine fluoride concentration has been stipulated ; nonetheless , there is scarce data regarding df prevalence and urinary fluoride excretion in adolescents living in rural communities with no central water supplies and where concentrations of fluoride may be above optimal . this descriptive study aimed to assess urinary fluoride concentration in a community where water supplies contain higher amounts of fluoride than recommended . this cross - sectional study was conducted in an endemic fluorosis area in guanajuato state where natural high concentrations of fluoride in groundwater and endemic dental fluorosis have been reported . areas with the highest concentration of fluoride are located toward the northwest between san felipe , san luis de la paz , and dolores hidalgo . participants were individuals aged 1120 years who were born and had resided in the area since their birth . a questionnaire was administered to all the participants at the time of admission to collect demographic data . fejerskov index ( tfi ) , which was selected because of its accuracy to identify df severity . one trained examiner performed clinical evaluations ; previously intraexaminer reliability was assessed using the kappa test ( kappa value = 0.82 ) . anthropometric measures such as weight and height were registered , and using these data body mass index ( bmi ) was calculated . percentage of body fat was also assessed by the bioimpedance method obtained by using a tanita scale sc240 . early morning spot urine sample were recollected in polyethylene containers and stored at 20c until analysis . urine fluoride ( uf ) concentrations were determined using an electronic meter ( orion 720a ) and a fluoride - specific ion electrode , which was calibrated with fresh , serially diluted standard solutions . during the measurement , written informed consent was obtained from all the participants or by their legal guardians in case they were minors . this study was approved by the committee of ethics the national autonomous university of mexico , enes len . descriptive analysis of the data ( arithmetic mean , standard deviation and percentages ) were obtained , bivariate analyses were performed to compare variables , and then a logistic regression model was created . population was divided into two groups according to the presence or absence of severe fluorosis ( tfi < 6 vs tfi > 6 ) . data was processed using spss version 21 for windows ( statistical package for the social sciences , spss inc . a total of 307 participants were included ; 59.9% ( n = 184 ) were females and 40.1% ( n = 123 ) were males . fluoride content in urine ranged between 0.5 and 6.65 mg / l , with a mean of 1.27 1.2 mg / l [ table 1 ] . descriptive data about age and urine fluoride concentration by sex most of the population ( 62.5% ) showed normal weight ; 21.5% were underweight , 11.1% were overweight , and 4.9% were obese . df was present in 91.9% of the participants , of which 61.6% were ( tfi > 4 ) moderate or severe cases , as observed in table 2 . teeth more frequently affected were premolars and those less affected were central inferior incisors . nutritional status and dental fluorosis severity by sex bivariate tests were performed to compare uf concentration according to different variables such as sex , bmi , and tfi . according to the kolmogorov smirnov test , the distribution of data was not normal and hence nonparametric tests were used . no differences in uf concentrations among girls and boys were observed ( mann whitney u test = 10589.50 , p = 0.335 ) . regarding bmi and uf , no difference in the prevalence of df according to nutritional status was observed ( chi square test = 29.746 ; p = 0.326 ) . nonetheless , significant differences ( chi square test = 11.22 ; p = 0.011 ) were observed when comparing the prevalence of severe df ( tf 6 ) and nutritional status ; 42% of the children having tfi 6 were underweight while of those having tfi < 6 18.6% were underweight , as shown in table 3 . urine fluoride concentration and dental severe fluorosis according to nutritional status positive correlation was observed among urine fluoride concentration and fluorosis severity ( rho spearman = 0.224 ; p < 0.001 ) . to perform bivariate analysis to identify the association between uf concentration and dental fluorosis severity status , this last variable was recoded in order to reduce it into 5 categories ; significant differences were noted ( kruskal wallis test = 16.200 ; p = 0.003 ) . children having dental fluorosis tfi = 79 had greater concentration of fluoride in urine [ table 4 ] . urine fluoride concentration according to dental fluorosis severity a logistic model was constructed to explore the association between severe dental fluorosis ( tfi < 6 vs tfi 6 ) and fluoride concentration in urine , controlled by age , sex , nutritional status , ( bmi ) and body fat percentage . it was observed that sex , age , body fat , and uf concentration were variables that were statistically significant in this model . male gender ( or = 0.127 ) , increment of percentage in body fat ( or = 0.875 ) and age ( or = 0.640 ) were protective factors . the increment of uf ( or = 1.40 ) concentration was a related risk to have severe df [ table 5 ] . potos state , in an area where fluoride level was similar ( 4.54 ppm ) , df was present in all participants , of which 95% had severe cases . in a mexican community where fluoride concentration was lower ( 1.9 ppm ) than that found in our study area , df prevalence was 98% , being severe in 47% . mexico found df in 85.5% of the population when fluoride in water was 3.005.99 ppm . ambient temperature , meters above mean sea level , risk practices as direct consumption of boiled water , and preparing food with tap water may explain the differences in df prevalence , even when concentration of fluoride in water are similar . range of fluoride content in urine was similar to that reported in other mexican children population , aged 6 - 12 , authors reported a range of uf concentration of 11.1 to 5.9 mg / l ; with a mean of uf content of ( 3.14 1.09mg / l . in an indian group aged 6 to 18 , the highest uf concentration recorded was 17 in other study in indian population , in individuals aged 1116 years , fluoride concentration found in urine samples ranged from 0.90 to 3.25 mg / l with an average of 2.35 mg / l . these variations might derived not only from variation on water fluoride content but from different use and consumption practices of water and other sources of fluoride among populations . we observed a positive correlation among uf concentration and fluorosis severity ; these results are congruent with those reported by jarqun - yaez et al . who found that urine fluoride concentration was more elevated in those showing greater fluorosis severity . they reported levels of 2.66 ( 0.89 ) in children with tfi of 45 , 3.11 ( 1.06 ) in the tfi of 67 , and 3.75 ( 1.10 ) ppm in tfi of 89 . nonetheless , heintze et al . reported no correlation between uf levels and df , however , that study was conducted in low - fluoride areas , which may be one of the causes of these different results . age was significantly associated with df , suggesting that age is a protective factor ; nonetheless , this does not imply that df decreases as age . increases , but it is not possible because df is irreversible . rather , these results might suggest that the problem is exacerbating , probably reflecting an increase in the consumption of fluoride in new generations that could come from higher concentration of fluoride in the water of the zone , as suggested by some authors who reported that as the depth of water extraction increases the concentration of this element also increases , raising the risk of developing df . pontigo - loyola et al . also reported similar results showing that children aged 12 years had greater chance to have fluorosis compared to those aged 15 years . the epidemiology system for oral diseases ( sivepab ) in 2010 also proposed an increase in df prevalence , especially in the younger age groups ( under 25 years ) . similarly , this has been observed in other countries , for instance , in a study performed in rural areas of brazil was observed that children between 10 and 12 years and those between 13 and 15 years of age had greater chance of having severe dental fluorosis in comparison with the younger children and individuals aged between 16 and 22 years . these results contrast to the study by rwenyonyi et al . who found significant increase in the severity of fluorosis with increasing age in a community with high concentration of fluoride in water . on the other hand reported that overall fluoride exposure dose has negative correlation with bmi ( r = 0.083 ) , which would be similar to that found in our population . we found no difference in the prevalence of df according to nutritional status , nonetheless significant differences were observed when comparing the prevalence of severe df , with 42% of the children having tfi 6 being underweight . some epidemiological studies have indicated that manifestations of fluorosis are more marked among communities exposed to chronic malnutrition . showed that , among participants with poor nutrition , the prevalence of df increased to 61.6% and skeletal fluorosis increased to 23.9% . furthermore in a study by irigoyen et al . in mexico , association between malnutrition and defects in the enamel were observed in an area where the water contained 2.7 mg / l of fluoride . similarly prez - prez et al . observed statistically significant differences in height for age , and reported a or = 2.66 for children with short stature to present fluorosis tf 4 . in addition , it was observed that males have less risk to present severe df than girls , these results are congruent to those reported by ramezani et al . who observed greater prevalence of severe fluorosis in girls ( 65% ) than in boys ( 34.2% ) . fluoride concentration in the water of this population exceeds the permissible limits for human consumption ( 0.71.5mg / l ) ; owing to the potential of adverse health effects of this situation , immediate actions are needed to reduce the exposure , thus diminishing adverse health effects in this population as df , which is an irreversible alteration . hence , actions taken would not only reduce dental fluorosis in future generations but also would prevent the prevalence or severity of other alterations that excessive consumption of fluoride can cause . most of the studied population had df and most of the cases were moderate or severe . water fluoride concentration of this population exceeds the limits stipulated by national and international norms . given the potential of adverse health effects that this may produce , immediate actions are needed to reduce the exposure to this element .

objectivethere was no abundance of data on the use of anticoagulant in patients with previous high risk of thromboembolic conditions under a newly developed intracranial hemorrhage in korean society . the purpose of this study was to evaluate the safety of discontinuance and suggest the proper time period for discontinuance of anticoagulant among these patients.methodswe reviewed the medical records of 19 patients who took anticoagulant because of thromboembolic problems and were admitted to our department with newly developed anticoagulation associated intracranial hemorrhage ( aaich ) , and stopped taking medicine due to concern of rebleeding from january 2008 to december 2012 . analysis of the incidence of thromboembolic complications and proper withdrawal time of anticoagulant was performed using the kaplan - meier method.resultsour patients showed high risk for thromboembolic complication . the cha2ds2-vasc score ranged from two to five . thromboembolic complication occurred in eight ( 42.1% ) out of 19 patients without restarting anticoagulant since the initial hemorrhage . among them , three patients ( 37.5% ) died from direct thromboembolic complications . mean time to outbreak of thromboembolic complication was 21.3814.89 days ( range , 8 - 56 days ) . the probability of thromboembolic complications at 7 , 14 , and 30 days since cessation of anticoagulation was 0.00 , 10.53 , and 38.49% , respectively.conclusionshort term discontinuance of anticoagulant within seven days in patients with aaich who are at high embolic risk ( cha2ds2-vasc score > 2 ) appears to be relatively safe in korean people . however , prolonged cessation ( more than seven days ) may result in increased incidence of catastrophic thromboembolic complications . objective : the aim of this study was to assess urine fluoride concentration , nutritional status , and dental fluorosis in adolescent students living in the rural areas of guanajuato , mexico.materials and methods : a cross - sectional study was conducted including participants aged 1120 years . the presence and severity of dental fluorosis was registered according to the thylstrup and fejerskov index ( tfi ) criteria . anthropometric measures were also recorded . urine sample of the first morning spot was recollected to assess urine fluoride concentration by using the potentiometric method with an ion - selective electrode . water samples were also recollected and analyzed . bivariate tests were performed to compare urine fluoride concentration according to different variables such as sex , body mass index , and tfi . nonparametric tests were used . a logistic regression model was performed ( spss 21.0).results : this study included 307 participants with a mean age of 15.6 1.6 ; 62.5% of the participants showed normal weight . a total of 91.9% of the participants had dental fluorosis , and 61.6% had tfi > 4 . mean fluoride content in urine ranged between 0.5 and 6.65 mg / l , with a mean of 1.27 1.2 mg / l . underweight children showed greater urine fluoride concentration . the increment of urine fluoride was a related ( or = 1.40 ) to having severe dental fluorosis.conclusions:most of the studied population had moderate or severe dental fluorosis . urine fluoride concentration was related to fluorosis severity and nutritional status . underweight children showed greater urine fluoride concentration as well as severe dental fluorosis .

solid organ transplantation provides an effective therapy for patients with kidney , liver , heart , and pulmonary failure . long - term graft survival is limited by adaptive alloimmune responses directed against transplant ( typically allogeneic major histocompatibility complex [ mhc ] ) antigens , that are expressed within the organ and on endothelial cell surfaces and that interface with circulating recipient immune cells . in addition , it is appreciated that a substantial number of memory t cells reside within non - lymphoid tissues ( mueller et al . , 2013 , solid organ allografts may therefore deliver passenger donor lymphocytes to the recipient after transplantation . currently , little is known about whether passenger lymphocytes remain in the allograft or reach recipient secondary lymphoid organs or how long they survive , given that their likely recognition by natural killer ( nk ) cells might be expected to ensure rapid elimination . however , the precise role of nk cells in solid organ transplantation remains unclear ( gill , 2010 , hadad et al . , 2014 , van der touw and bromberg , 2010 , hidalgo et al . , 2010 ) , and early transplant studies indicate that circulating donor lymphocytes are often detectable in human transplant recipients , albeit in small numbers ( starzl et al . their presence may manifest as devastating , acute graft - versus - host ( gvh ) disease ( sharma et al . , 2012 ) , or as passenger lymphocyte syndrome , in which hemolysis is triggered by donor b cell recognition of mismatched abo blood group antigens in the recipient ( nadarajah et al . , 2013 ) . thus , the impact of passenger lymphocytes on the recipient immune response to the allograft has still to be clarified ( turner et al . , 2014 ) . we have shown that in a murine heart transplant model with an isolated mhc class ii - mismatch [ b6(c)-h2-ab1bm12/khegj ( bm12 ) to c57bl/6 ( b6 ) ] , passenger bm12 cd4 t cell recognition of i - a mhc class ii on host b cells triggers the production of anti - nuclear autoantibody , which causes allograft vasculopathy ( motallebzadeh et al . , 2012 , for example , activation of host dendritic cells ( dcs ) and macrophages following recognition of surface mhc class ii by donor cd4 t cells could prompt more vigorous host alloimmunity from more effective processing and presentation of graft alloantigen as self - restricted peptide fragments . to examine the possibility that passenger donor lymphocytes augment conventional host alloimmunity , we developed a murine transplant model incorporating a new bm12-derivative donor strain that expresses additional mhc class i and class ii alloantigens to act as targets for conventional cellular and humoral allorecognition ( ali et al . , 2016 ) . here we describe how in this model , heart allografts provoke autoantibody production in b6 recipients as a consequence of gvh recognition by passenger donor cd4 t cells . we show that even though donor cd4 t cells survive for only a few days after heart transplantation , their survival provokes a marked and long - lasting augmentation of cellular and humoral alloimmunity and results in early allograft rejection . however , this augmentation is prevented in completely mismatched strain combinations by rapid nk cell killing of donor lymphocytes . these data have important clinical implications , suggesting that partial mhc mismatch between donor and recipient to promote nk cells responses against passenger lymphocytes may reduce alloimmune responses . human organs procured for transplantation , including kidney , liver , and heart , contain significant populations of effector and effector - memory cd4 and cd8 t lymphocytes ( figure s1 ) . we therefore sought to examine the impact of these passenger donor lymphocytes on recipient adaptive alloimmune responses . to address this question , we developed a mouse strain that expressed multiple mhc alloantigens , sufficient to stimulate cellular and humoral alloimmunity , in addition to provoking humoral autoimmunity . a series of backcrosses were performed between bm12 , b6.k ( honjo et al . , 2004b ) , and b6.i - e ( conlon et al . , 2012a ) strains to derive the bm12.k.ie strain , which differs from the b6 recipient strain at the classical mhc class i k and class ii a and e loci ( h-2 , k , a , e , and d ; figures 1a and s2 ) . when bm12.k.ie hearts allografts were transplanted into b6 recipients , the additional mhc class i h-2k and class ii i - e mismatched alloantigens provoked strong alloimmune responses , with production of long - lasting alloantibody to both antigens ( figure 1b ) . recipients also developed anti - nuclear autoantibody ( figure 1c ) that was comparable in magnitude to the responses previously observed in b6 recipients of bm12 heart allografts ( win et al . these antibody responses were associated with c4d complement deposition on heart graft endothelium ( figure 1d ) , which was not evident in syngeneic heart transplants , suggesting a humoral component to the allograft vasculopathy that developed within allografts by day 100 ( figure 1e ) . to determine whether , as in the bm12 to b6 model , autoantibody production in b6 recipients of bm12.k.ie heart allografts was due to donor cd4 t cell allorecognition of recipient i - a mhc class ii ( callaghan et al . bm12.k.ie donor mice were treated with depleting anti - cd4 monoclonal antibody ( mab ) before sacrifice . this resulted in profound depletion of circulating and tissue resident cd4 t cell compartments by the time of heart allograft procurement ( figures 1f and 1 g ) . anti - cd4 antibody was not carried over to the recipient ( figure 1h ) ; nevertheless , donor treatment with anti - cd4 mab abrogated the recipient autoantibody response ( figure 1i ) , confirming that passenger cd4 t cells within the bm12.k.ie donor heart are responsible for initiating recipient humoral autoimmunity . despite the development of humoral autoimmunity , no overt autoimmune disease was observed in kidney , liver , skin , or native heart in b6 recipients up to 100 days after transplantation with a bm12.k.ie heart allograft ( figure s3 ) . the ability , through specific targeting of the donor cd4 t cell population , to independently manipulate recipient autoimmune and alloimmune responses provided a means to examine whether gvh allorecognition augments host alloimmunity . comparison of recipient cellular and humoral alloimmune responses in recipients of unmodified and cd4 t cell - depleted bm12.k.ie heart allografts revealed that alloantibody responses against the h-2k alloantigen were substantially reduced in recipients of cd4 t cell - depleted allografts ( figure 2a and s4 ) . responses against the disparate donor mhc class ii alloantigen were similarly ameliorated ( figure 2b ) . allo- and autoantibody responses were restored in recipients of cd4 t cell - depleted bm12.k.ie heart allografts by adoptive transfer of purified donor cd4 t cells at transplantation ( figures 1i and 2a ) . the disparate h-2k alloantigen might be expected to act as a target for recognition by recipient cytotoxic cd8 t cells ( harper et al . , 2015 ) , but whereas b6 recipients of fully mhc - mismatched balb / c heart allografts generated robust cd8 t cell responses , the response in recipients of unmodified bm12.k.ie heart allografts was weak and transient ( figure 2c ) . nevertheless , cytotoxic cd8 t cell responses were barely detectable in recipients of cd4 t cell - depleted bm12.k.ie heart transplant recipients at any time point ( figure 2c ) . helper cd4 t cell alloresponses were also examined in the recipient groups , by evaluating proliferation of tcr75 cd4 t cells that were adoptively transferred 5 weeks after the heart transplant . tcr75 cd4 t cells recognize k alloantigen via the indirect pathway ( ali et al . , 2013 ) as self - i - a - restricted , but not donor - i - a - restricted , allopeptide ( honjo et al . , 2004a , conlon et al . , 2012b ) . in recipients of cd4 t cell - replete heart grafts , marked tcr75 t cell proliferation was observed , indicating ongoing presentation of immunogenic k allopeptide epitope . in contrast , tcr75 t cell responses in recipients of cd4 t cell - depleted hearts were approximately 50% weaker ( figure 2d ) . the marked reduction in the alloimmune response to cd4 t cell - depleted bm12.k.ie heart allografts ameliorated graft rejection , in that vasculopathy was minimal in heart allografts from cd4 t cell - depleted donors and comparable to that observed in syngeneic heart transplants ( figure 3a ) . in addition , all heart transplants from cd4 t cell - depleted donors were beating strongly at day 50 ( figure 3b ) . adoptive transfer of donor cd4 t cells at time of transplant to recipients of cd4 t cell - depleted bm12.k.ie heart allografts restored the development of allograft vasculopathy ( figure 3a ) . in contrast to human organs where memory t cell populations dominate ( figure s1 ) , the cd4 t cell compartment in mice housed in specific - pathogen - free conditions is maintained in a largely naive state . we therefore sought to examine how memory cd4 t cells within an allograft might influence host alloimmunity , by priming bm12.k.ie donors with a b6 skin graft 6 weeks before procurement of the heart allograft to generate resident memory t cells . heart allografts from such donors were rejected more rapidly by b6 recipients than were heart grafts from naive donors , and they triggered augmented auto- and alloantibody responses ( figures 3a3d ) . amplification of the recipient alloreactive t - b lymphocyte axis is likely the principal mechanism by which donor cd4 t cell gvh recognition triggers accelerated graft rejection , because host germinal center ( gc ) alloantibody responses were less established in recipients of t cell - depleted , than t cell - replete , heart allografts ( figure 3e ) , as was complement c4d deposition on allograft endothelium ( figure 1d ) . similarly , heart grafts were not rejected , and developed only minimal vasculopathy , when transplanted into either b cell - depleted ( figure s5 ) or t cell - deficient tcrbd recipients ( figures 3a and 3b ) . to examine whether the augmentation in recipient cd4 t cell responses triggered by donor gvh recognition was dependent upon host b cell immunity , b cell - depleted recipients were transplanted with either cd4 t cell - replete or t cell - deficient bm12.k.ie heart grafts , and proliferation of transferred tcr75 t cells was examined as earlier . for recipients of cd4 t cell - replete heart grafts , t cells was substantially less in b cell - depleted than in untreated recipients ( figure 2e ) and approximated that observed in untreated recipients of cd4 t cell - depleted bm12.k.ie heart allografts . furthermore , unlike b cell - replete recipients , proliferation of transferred tcr75 cd4 t cells in b cell - depleted recipients was not influenced by depletion of donor cd4 t cells ( figure 2e ) . the role of recipient b cells in gvh - mediated augmentation of recipient t cell alloreactivity does not simply reflect function as the major cell population expressing target i - a for optimal gvh activation of donor cd4 t cells , because the latter still divided readily in b cell - depleted donors ( figure 3f ) , such that no carboxyfluorescein succinimidyl ester ( cfse ) staining was detectable in the sub - population of alloreactive bm12.k.ie cd4 t cells as early as 3 days after transfer ( figure 3f ) . although transplantation of bm12.k.ie hearts into tcrbd recipients prompted autoantibody and weak alloantibody responses ( figures 3 g and 4h ) , there was no associated gc activity ( figure 3e ) . thus , the requirement for host cd4 t cells in bm12.k.ie heart graft rejection appears to reflect provision of essential help for development of sophisticated host humoral alloimmunity , a function not provided by donor cd4 t cells ; transferred donor cd4 t cells are rapidly killed by adaptive alloimmune recognition in b6 hosts , because whereas bm12.k.ie cd4 t cells were readily identified 7 days after transfer into rag-2 hosts , they were undetectable following transfer into wild - type b6 hosts ( figure 3i ) . hence , it is unlikely that the donor cd4 t cells survive long enough to contribute directly to the progression of allograft vasculopathy . their effect appears to be mediated principally through a relatively short - lived interaction with host b cells , but prolonged augmentation of humoral alloimmunity is dependent upon additional help from host cd4 t cells . in considering how donor cd4 t cells amplify humoral alloimmunity , transfer of bm12 cd4 t cells into b6 hosts prompted upregulation of mhc class ii expression on mature b cells ( figure 4a ) , in keeping with global activation from recognition of all allogeneic mhc class ii complexes on their surface . yet only a limited repertoire of antibody directed against nuclear self - antigen was produced ( data not shown ) . to examine the hypothesis that plasma cell differentiation requires b cell receptor ( bcr ) ligation , in addition to cognate interaction between the mhc class ii complex and the donor cd4 t cell , tcrbd b6 mice were challenged with purified bm12 cd4 t cells and immunized with ovalbumin ( ova ) protein . in this situation , cd4 t cell help for humoral responses can only be provided by the transferred donor cd4 t cells . as expected , mice in both groups developed anti - nuclear autoantibody , but anti - ova immunoglobulin g ( igg ) responses were only detectable in the group immunized with ova ( figures 4b and 4c ) . similarly , challenge of tcrbd b6 mice with cd4 t cells from bm12 mice that expressed transgenic h-2k antigen ( bm12.k ) provoked autoantibody , but also strong anti - k igg alloantibody , which was not observed in tcrbd b6 mice challenged with bm12 cd4 t cells ( figures 4d and 4e ) . bm12.k cd4 t cells are selected against reactivity to self ( i - a)-restricted k peptide and are unable to provide help to k - specific bm12 b cells for generating anti - h-2k antibody ( figures 4f and 4 g ) . thus , their provision of help for generating anti - h-2k antibody in b6 hosts reflects peptide - degenerate direct - pathway allorecognition of i - a mhc class ii on h-2k - specific b6 b cells that , with simultaneous bcr ligation , provokes class - switched alloantibody . these alloantibody responses presumably explain why bm12.k.ie cd4 t cells are undetectable within a week of transfer into b6 tcrbd mice but survive long term in rag-2 mice ( figure 3i ) . in summary , despite being tolerant of h-2k antigen on the bm12 background , bm12.k cd4 t cells provoke anti - k alloantibody when transferred into b6 hosts ; this alloantibody results in rapid destruction of the bm12.k cd4 t cells . whether the amplification of host humoral immunity by gvh recognition is an intrinsic component of the alloresponse or is dependent upon the degree of mhc mismatch between donor and recipient has not been addressed . given that donor bm12.k.ie cd4 t cells survive long term in rag-2 b6 hosts ( figure 3i ) , we examined whether innate immune evasion , and specifically lack of nk cell allorecognition of donor lymphocytes , was critical in triggering autoantibody generation . in this regard , cd4 t cells purified from the completely mismatched balb / c donor strain did not survive when injected into b6 rag-2 hosts and did not provoke humoral auto- or alloimmunity upon injection into b6 tcrbd mice ( figures 5a5c ) . this contrasts with long - term survival and development of strong igg allo- and autoantibody when purified cd4 t cells from the less mismatched strains were injected ( figures 5a5c ) . furthermore , depletion of nk cells by administration of anti - nk1.1 antibody , in the b6 rag-2 recipients , resulted in long - term survival of transferred balb / c cd4 t cells and , in tcrbd recipients , provoked class - switched auto- and alloantibody responses ( figures 5b5d ) that were even stronger than those observed upon administration of cd4 t cells from the less mismatched donor strains . nk t cells , which also express nk1.1 , do not develop in tcrbd mice ( figure s6 ) ; these experiments therefore serve as an apposite control that the administered anti - nk1.1 antibody is acting principally upon nk cells . irrespective of gvh recognition , injection of balb / c cd4 t cells into wild - type , immunocompetent b6 mice would be expected to provoke alloantibody , but autoantibody was only produced if host nk cells were depleted simultaneously ( figure 5e ) , confirming that elimination of the transferred donor cd4 t cell population , by either host cytotoxic cd8 t cell or alloantibody responses , does not occur quickly enough to obviate a gvh response and that nk cell allorecognition is instead essential for its prevention . these observations suggest that gvh - mediated amplification of host alloimmune effector responses is normally prevented in mhc - mismatched transplant models by host nk cell alloreactivity . the role of nk cells in rejection of completely mismatched balb / c hearts by b6 recipients was therefore examined . however , in this model of acute rejection , unmodified b6 recipients reject balb / c heart allografts within days , and it seemed unlikely that host nk cell depletion would influence such a robust rejection response . instead , a further model of chronic alloantibody - mediated allograft vasculopathy was developed in which b6 tcrbd recipients of balb / c heart allografts are reconstituted at transplantation with b6 tcr75 cd4 t cells but at limiting numbers ( 10 per mouse ) , such that rejection occurs slowly and is mediated by anti - h-2k gc alloantibody responses , with help provided by differentiation of the transferred tcr75 t cells to follicular helper t cells ( figure 6a ) . in contrast to the gradually evolving anti - k alloantibody responses observed in nk cell - replete recipients , responses in the nk cell - depleted recipients were stronger ( figure 6b ) , and the heart grafts were rejected within the first week ( figure 6c ) . autoantibody was also generated in the nk cell - depleted recipients ( figure 6d ) , confirming the development of gvh responses mediated by donor balb / c cd4 t cells . critically , autoantibody generation , the augmented alloantibody response , and rapid allograft rejection were ameliorated in nk cell - depleted recipients by depletion of cd4 t cells from the balb / c donor before heart graft procurement ( figures 6b6d ) . finally , to test the relevance of our findings to a model in which graft rejection is prevented by administration of immunosuppression , as occurs routinely in clinical practice , heart allografts from balb / c donor mice that had been challenged 6 weeks earlier with a b6 skin graft were transplanted into b6 recipients that were treated with anti - cd154 monoclonal antibody at transplantation . in b6 recipients of heart grafts from unmodified donors , this protocol results in long - term allograft survival ( larsen et al . , 1996 , ali et al . , 2016 ) , without development of autoantibody ( figure 6e ) , but in recipients of heart allografts from challenged donors ( that contained memory passenger cd4 t cells ) , depletion of nk cells at transplantation resulted in development of anti - nuclear autoantibody and more pronounced splenic gc activity ( figures 6e and 6f ) . despite the robust gc response , anti h-2k alloantibody responses were not observed ( data not shown ) . depletion of cd4 t cells in the donor before heart allograft procurement abrogated the autoantibody response ( figures 6e and 6f ) . although the presence of donor lymphocytes in the circulation of recipients of solid organ allografts was first demonstrated more than 2 decades ago ( starzl et al . , 1992a , starzl et al . , 1992b ) , the extent to which they affect recipient alloimmunity has remained unclear . clarification of the contribution of passenger donor lymphocytes to graft rejection has become more pertinent with the realization that non - lymphoid tissue contains substantial populations of either resident or circulating memory t lymphocytes , and their presence has been described within all solid organs currently transplanted in humans ( casey et al . , 2012 , sathaliyawala et al . , 2013 ) . here , we used a combination of donor cd4 t cell depletion and adoptive transfer of donor cd4 t cells , in conjunction with transplantation of heart allografts from primed donors , to demonstrate that gvh allorecognition by donor cd4 t cells augments recipient alloimmunity and that this augmentation is more pronounced for allografts procured from donors sensitized against recipient mhc . our findings thus reveal a mechanism by which donor lymphocytes may influence graft rejection and suggest that their impact may be more important than previously considered . pivotal to this augmentation of host alloimmunity is the ability of donor cd4 t cells to recognize host mhc class ii via the direct pathway ( ali et al . , 2013 ) this provides an unusual form of peptide - degenerate help , reflecting the unique nature of direct - pathway allorecognition ( macdonald et al . , 2009 , ali et al . , 2013 ) , in which the precursor frequency of cd4 t cells that respond to a particular mhc class ii alloantigen is 100- to 1,000-fold greater than for the response against conventional , self - restricted peptide antigen , because all mhc class ii alloantigen complexes are recognized as foreign , irrespective of bound peptide . this results in activation of all recipient b cells , but we detail that differentiation to an igg antibody - secreting plasma cell is dependent upon simultaneous b cell receptor ligation . thus , although donor cd4 t cells can provide help to recipient b cells in an antigen - independent fashion , antigen specificity is maintained through the requirement for b cell receptor ligation . this atypical help does not , however , completely replicate conventional cognate help provided by cd4 t cells with self - restricted specificity for peptide derived from target antigen , because although gvh recognition by donor cd4 t cells could trigger auto- and alloantibody responses , these were not sustained in the absence of a recipient cd4 t cell population , and allograft rejection did not occur . our findings thus reveal an interaction between donor and recipient t and b lymphocytes , as depicted in figure 7 . passenger lymphocytes only augmented recipient alloimmunity if they were not eliminated rapidly by recipient nk cells . the contribution of nk cell alloresponses to allograft rejection is still debated ( gill , 2010 , hadad et al . , 2014 , van der touw and bromberg , 2010 ) . it has been suggested that host nk cells promote allograft rejection ( maier et al . , 2001 , uehara et al . , 2005 , 2008 ) either through the destruction of opsonized donor cells or perhaps through regulation of t cell immunity ( maier et al . , 2001 ) . our results suggest the converse : that a major function of nk cells is inhibition of destructive cellular and humoral alloimmunity that is triggered by passenger cd4 this accords with several studies reporting a key role for nk cells in allograft tolerance ( beilke et al . , 2005 , yu et al . , 2006 ) . the mechanisms by which nk cells promote tolerance in these studies have not been firmly established , but inhibition of recipient alloimmune responses through killing of donor dcs may be important ( yu et al . , 2006 ) ; in support , laffont et al . ( 2008 ) have reported that nk cell - mediated destruction of donor dcs downregulates cd4 t cell alloimmunity . the different mechanism highlighted by our study the killing of passenger lymphocytes may be more clinically relevant , because irrespective of nk cell allorecognition , adaptive alloimmune recognition would be expected to result in prompt destruction of donor dcs ; in the laffont et al . ( 2008 ) study , cd8 mice were studied to obviate rapid killing by cytotoxic t lymphocytes . our results reveal that the critical window for passenger donor cd4 t cells to augment host alloimmunity is within the first few days after transplant and that evasion of nk cell - mediated killing is essential for this effect . donor cd4 t cells prompt host adaptive responses that engender their own rapid destruction , but the delay in development of these responses , at most a few days when compared to nk cell recognition , is sufficient for gvh recognition to occur . one could argue , on the basis of the derived nature of the bm12.kd.ie donor strain and the lack of requirement for administration of immunosuppression , that the clinical relevance is limited . similarly , it is perhaps surprising that passenger donor lymphocytes were present in sufficiently large numbers within heart allografts to provoke such a marked augmentation in the host s alloimmune response . against this , cd4 t cells could be readily detected in all sampled human organs that have been procured for transplantation but not used . in addition , to counter concerns regarding the wider applicability of the bm12.kd.ie model , we employed an additional model of chronic allograft vasculopathy using completely mismatched balb / c donor and b6 recipient strains . this model enabled clarification of the crucial role of host nk cells in killing donor hematopoietic cells ; nevertheless , the potential for passenger donor lymphocytes to augment host alloimmunity was again observed . we further demonstrated in this model that memory donor cd4 t cells ( as would be expected to be present within human allografts ) were able to provide co - stimulation - independent help to naive recipient b cells for production of a gc autoantibody response . with regards the seemingly large numbers of donor lymphocytes contained within our murine heart allografts , our experiments were not able to distinguish whether these were truly resident within the parenchyma or trapped within the microcirculation of the heart allograft , and it is possible that different procurement and storage techniques used in clinical transplantation denude a heart allograft of most of its passenger lymphocyte populations . against this , deliberate flushing of our mouse hearts via the coronary arteries at explant ( as typically occurs in clinical heart allograft procurement ) did not make any appreciable difference to the numbers of cd4 t cells subsequently found within the heart allograft ( data not shown ) . in any event , we stress that the heart allograft model was used as a means of delineating the precise mechanism by which donor cd4 t cells influence the host s response to an allograft . in this respect , whereas the impact of passenger lymphocytes in clinical cardiac transplantation may be limited , lung , small bowel , and composite tissue allografts will almost certainly transfer large numbers of donor lymphocytes that originate from organized lymphoid tissue contained within the allograft . transplant outcomes for these organs are poorer than for other organs ; for example , a report of chronic face allograft rejection described the development of autoimmune , scleroderma - type features consistent with skin manifestations of chronic gvh ( petruzzo et al . , 2015 ) . one might therefore predict that transplant outcomes would be particularly poor for individuals that receive such organs from donors matched for killer cell immunoglobulin - like receptor ( kir ) recognition ( which occurs in approximately 50% of kidney transplant pairings ; van bergen et al . , 2011 ) , because the avoidance of immediate host nk cell detection would enable passenger donor lymphocytes to potentiate host alloimmunity . however , the impact of nk cell alloreactivity in transplant outcomes remains uncertain ( tran et al . , 2013 , van bergen et al . , 2011 ) , possibly because studies to date have avoided the confounding impact of human leukocyte antigen ( hla ) mismatches on allograft survival by including only hla - matched donor - recipient combinations , whereas our findings suggest that kir - ligand matching would compromise transplant outcomes when donor and recipient are mismatched additionally at the hla class ii loci . it is likely that the ability of donor cd4 t cells to provide peptide - independent help to host b cells has implications beyond solid organ transplantation . in hematopoietic stem cell transplantation , an association between chronic gvh disease and humoral immunity is increasingly recognized ( nakasone et al . , 2015 , dubovsky et al . , 2014 , shimabukuro - vornhagen et al . our findings suggest that this may relate to a chimeric state in which the co - existence of populations of donor and recipient t and b lymphocytes tends to provoke destructive alloantibody responses . in addition , persistence of a mixed chimeric state implies that reciprocal nk cell tolerance to donor and host had been achieved ( narni - mancinelli et al . , 2013 ) , which may be particularly relevant to strategies for hematological malignancy that use less toxic , non - myeloablative conditioning to initially establish mixed hematopoietic chimerism and then later convert to full donor chimerism by infusion of donor lymphocytes ( chang and huang , 2013 ) . our findings suggest that inhibition of host nk cell alloresponses may enable gvh recognition by cd4 t cells within the subsequent donor infusion to provide promiscuous help for antibody production from residual host b cells that are concurrently engaging target antigen . this may explain reports documenting the development of humoral immunity against tumor antigen following establishment of mixed hematopoietic chimerism ( bellucci et al . similarly , the presence of donor cd4 t cells within donor lymphocyte infusions has been associated with loss of donor mixed hematopoietic chimerism ( kim et al . , 2004 , hock et al . , 2014 ) , but rather than this being a bystander consequence of the general inflammatory milieu created by the gvh response ( hock et al . , 2014 ) , our results suggest that the loss may instead be due to cognate recognition of mhc class ii on the surface of recipient alloreactive b cells by donor cd4 t cells . in summary , we demonstrate an unexpected role for donor passenger cd4 t cells within allografts in the provision of help to recipient b cells for generating humoral responses directed against the transplant . passenger donor lymphocytes may therefore influence recipient alloimmune responses more profoundly than previously considered and represent a therapeutic target in solid organ transplantation . b6 ( h-2 ) and balb / c mice ( h-2 ) were purchased from charles river laboratories . bm12 mice and t cell receptor - deficient mice ( h-2 , tcrbd ) b6.129p2-tcrbtcrd tcr - transgenic rag-1 tcr75 mice ( h-2 ) , specific for i - a - restricted h-2k5468 peptide ( honjo et al . , 2004a ) and b6-tg(k)rpb ( b6.k ) mice , which express the full sequence of h-2k ( honjo et al . , 2004b ) b6 mice that lack i - a but express i - e ( b6.i - e ; conlon et al . , 2012a ) all animals were maintained in specific pathogen - free facilities , and experiments were approved by the uk home office animal ( scientific procedures ) act of 1986 . fully vascularized cardiac allografts were transplanted intra - abdominally ( conlon et al . , 2012b ) . grafts were excised at predetermined time points after transplantation and stored at 80c or fixed in 10% buffered formalin . in certain experiments , heart allografts were retrieved from donor mice challenged with a recipient strain skin allograft 6 weeks earlier , or recipients were additionally injected intraperitoneally ( i.p . ) with 500 g anti - cd154 mab ( clone mr-1 ; be0017 - 1 ; bio x cell ) on days 2 and 0 in relation to transplantation , a protocol that prevents acute allograft rejection but that results in development of chronic allograft vasculopathy . bone marrow - derived dendritic cells ( bmdcs ) were prepared as described previously ( curry et al . , 2007 ) . briefly , bone marrow ( bm ) was flushed from femurs and tibias with hank s balanced salt solution ( invitrogen ) . cells were disaggregated by passing through a 40-m mesh , and bm cells cultured in six - well plates at 3 10/ml in 6-ml complete medium ( rpmi 1640 , 10% fetal calf serum [ fcs ] , 100 iu / ml penicillin , 100 g / ml streptomycin , and 2 mm l - glutamine ; invitrogen ) , supplemented with murine granulocyte - macrophage colony - stimulating factor ( peprotech ) at 20 ng / ml and recombinant murine interleukin-4 ( peprotech ) at 10 ng / ml . cells were maintained by replacing half the culture medium with fresh medium on alternate days . nonadherent cells were discarded on day 4 , and dcs were used on day 8 for flow cytometric analysis . anti - nuclear autoantibody responses were determined by hep-2 indirect immunofluorescence ( the binding site ) , as described previously ( callaghan et al . , 2012 ) , by incubating test sera on slides coated with hep-2 cells and detecting bound antibody with fluorescein isothiocyanate ( fitc)-conjugated goat anti - mouse igg ( star 70 ; serotec ) . for each test serum , photomicrographs were taken , and the intensity of staining was determined by integrated morphometric analysis using metamorph software . the fluorescence value was then derived by comparison with a standard curve , obtained for each assay by serial dilutions of a pooled hyperimmune serum that was assigned an arbitrary value of 1,000 fluorescence units . sera were collected from experimental animals weekly and analyzed for anti - i - e alloantibody ( at week 4 in the case of balb / c alloantibody ) by flow cytometric detection of binding to target cells . briefly , target b6.i - e and balb / c bmdcs were first blocked with anti - mouse cd16/cd32 ( clone 2.4g2 ; bd pharmingen ) and then incubated with serial dilutions ( 3-fold ) of heat - inactivated test serum for 30 min . bound alloantibody was detected with fitc - conjugated goat anti - mouse igg ( star 70 ; serotec ) , and cells were analyzed by flow cytometry . for each sample , the geometric mean - channel fluorescence was obtained and plotted against dilution , and the area under the curve ( auc ) was then calculated as a percentage of the auc of a standard of pooled hyperimmune sera . serum samples were collected from experimental animals weekly and analyzed for the presence of anti - h-2k igg alloantibody by elisa . in brief , 96-well elisa plates ( immulon 4hbx ; thermo scientific ) were coated with recombinant conformational h-2k at 5 g / ml in na2co3-nahco3 buffer ( ph 9.6 ) . plates were blocked with 1% marvel dried skimmed milk powder ( premier international foods ) , tripling serial dilutions of test sera added and bound igg antibody detected by incubating with biotinylated rabbit f(ab)2 anti - mouse igg ( star11b ; abd serotec ) and extravidin peroxidase conjugate ( sigma ) . sure blue substrate ( kpl ) was then added , the reaction was stopped by the addition of 0.2 m h2so4 , and the absorbance ( optical density 450 ) was measured in a fluostar optima plate reader ( bmg labtech ) . for each sample , an absorbance versus dilution curve was plotted , and the auc was calculated ( conlon et al . , 2012a ) . the auc of an experimental sample was expressed as the percentage of positive control ( pooled hyperimmune ) serum . in certain experiments , mice were additionally immunized with ova protein 100 g in incomplete freund s adjuvant subcutaneously . anti - ova antibody was assayed in a similar fashion , and performed on test sera 4 weeks after immunization , using an ova - specific elisa . cd8 t cell enzyme - linked immunospot was performed as described ( sivaganesh et al . briefly , purified cd8 t cells were mixed with irradiated balb / c stimulator splenocytes and added to multiscreen hts filtration system plates ( millipore ) that had been coated with anti - mouse interferon- ( ifn- ; bd pharmingen ) in 0.1 m bicarbonate buffer ( ph 9.6 ) . plates were incubated at 37c and 5% co2 for 20 hr , and after washing , spots were developed with biotinylated rat anti - mouse ifn- ( bd pharmingen ) , followed by streptavidin - horseradish peroxidase and the substrate , h2o2 , together with the 3-amino-9-ethylcarbazole color indicator . plates were read ( autoimmun diagnostika ) , and data were expressed as spot counts per 10 responder cd8 t cells for each well . antigen - presenting cell - conjugated anti - mouse cd4 ( rm4 - 5 ) , fitc - conjugated anti - mouse cd19 ( 1d3 ) , r - phycoerythrin ( pe)-conjugated anti - mouse cd90.1/thy1.1 ( clone ox-7 ) , pe - cy7-conjugated anti - mouse cd4 ( clone l3t4 ) , pe - conjugated anti - mouse h-2k ( sf1 - 1.1 ) , and fitc - conjugated anti - mouse i - a ( clone af6 - 120.11 ) were purchased from bd pharmingen . peripheral blood ( depleted of erythrocytes by incubating with 0.17 m nh4cl red cell lysis buffer ) and splenic single - cell suspensions were blocked with anti - mouse cd16/cd32 ( clone 2.4g2 ; bd pharmingen ) , before staining with the relevant antibodies and dead cell exclusion dye 7-aminoactinomycin d ( bd pharmingen ) . all cells were analyzed on a facscanto ii flow cytometer with facsdiva software ( bd biosciences ) . single - cell suspensions of splenocytes obtained from tcr75 mice were stained with 5 m cfse ( molecular probes ) in the dark for 5 min and then quenched with 5% fcs / pbs . cfse - stained splenocytes ( 2 10 to 5 10 ) were injected intravenously ( i.v . ) into recipient mice and spleens harvested 4 or 7 days later ; flow cytometry was performed using allophycocyanin - conjugated anti - cd4 plus pe - conjugated anti - cd90.1/thy1.1 to identify tcr75 t cells . proliferation of wild - type bm12.k.ie cd4 t cells in b6 hosts was assessed similarly , by analysis of csfe - staining 3 days after transfer of 5 10 cells , with the caveat that in contrast to transfer of a monoclonal population , analysis was restricted to the relatively small ( 5% ) alloreactive population nested within a large wild - type repertoire that did not undergo proliferation . formalin - fixed hearts were paraffin mounted and stained using h&e and weigert s elastin van gieson method to delineate the internal elastic lamina and the severity of allograft vasculopathy assessed morphometrically , as reported previously ( motallebzadeh et al . , 2012 ) . complement c4d deposition was assessed on 7-m cryostat sections of donor heart allografts explanted after 50 days by an avidin - biotin - peroxidase technique ( vector laboratories ) , using unconjugated rat anti - mouse c4 mab ( 16d2 ; abcam ) , as described previously ( win et al . , 2009 ) . gcs were quantified on 7-m cryostat sections of recipient spleens harvested 50 days following transplant by immunofluorescence staining of b220 b cells using rat anti - mouse b220 ( clone ra3 - 6b2 ; bd pharmingen ) detected with cy3-conjugated goat anti - rat igg ( clone 112 - 165 - 143 , jackson immunoresearch laboratories ) and peanut agglutinin ( pna ) gc b cells using fitc - conjugated pna ( vector laboratories ) , as described previously ( conlon et al . , 2012b ) . numbers of pna gc were expressed as a percentage of total ( b220 ) lymphoid follicles . donor mice were injected i.p . with 2 1.0 mg doses of depleting anti - cd4 mab ( yts 191.1 ; hybridoma from the european collection of animal cell cultures ) 6 days and 1 day before heart graft procurement . depletion of cd4 t cells ( typically > 99% ) was confirmed by flow cytometric analysis of peripheral blood . to confirm cardiac parenchymal cd4 t cell depletion , donor hearts were homogenized following incubation with collagenase digestion buffer , as described previously ( sivaganesh et al . , 2013 ) , with a single - cell suspension prepared by filtration through a 40-m nylon cell strainer . cd4 t cells were quantified by flow cytometry , with a mean of 5,137 cd4 t cells identified in an untreated donor heart . in certain experiments , recipients of cd4 t cell - depleted allografts were adoptively transferred i.v . 1 10 donor cd4 t cells ( purified with anti - mouse cd4 microbeads ( mitenyi ) using an automacs separator ( mitenyi ) . 250 g depleting anti - cd20 mab ( 18b12 ; gifted by cherie butts at biogen idec ) 7 days before and 14 days after transplantation ( ueki et al . , depletion of b cells was confirmed by flow cytometry of peripheral blood mononuclear cells ( pbmcs ) the day before heart transplantation . depletion of the recipient nk cell population was achieved by injecting i.p . 500 g depleting anti - nk1.1 ( pk136 ; hybridoma from the european collection of animal cell cultures ) 2 days and 1 day before transplant or cell transfer and three times weekly thereafter . depletion of nk cells was confirmed by flow cytometry of pbmcs the day before transplantation or transfer . adoptive transfer studies of purified b6 , balb / c , bm12.k , and bm12.k.ie cd4 t cells into b6 , tcrbd , and tcrbd.bm12 mice were performed by injecting i.v . 1 10 cells purified as earlier . two - way anova was employed for comparison of intensity of hep-2 fluorescence scores and anti - h-2k antibody levels . graft survival was depicted using kaplan - meier analysis , and groups were compared by log rank ( mantel - cox ) testing . i.g.h . , j.m.a . , and g.j.p . wrote the first draft of the paper . i.g.h . , j.m.a . , s.j.f.h . , e.w . , j.a . , m.c.n . , m.s.q . , r.m .- z . , k.s .- p . , e.m.b . , j.a.b . , m.r.c . , t.m.c . , and g.j.p . reviewed and edited the manuscript , approving the final version . in ovo electroporation and in situ hybridization was performed as in previous studies ( 6 ) , and at least three embryos were analyzed for each condition . rna probes were transcribed in vitro from cdna clones provided by bbsrc ( bmp7 : chest290n8 ; bapx1 : chest177b22 ; pitx2 : chest76c15 ) , except for the neurog3 cdna provided by anne grapin - botton and the bmp4 cdna provided by cliff tabin . probes for nkx6.1 and hes1 were used as in earlier studies ( 6,17 ) the xenopus noggin cdna was provided by richard harland , and a cooh - terminal cmyc epitope tag inserted by pcr . the mouse dnalk3 was a cooh - terminal truncated receptor ( encoding aa 1237 ) lacking the kinase domain . a similar construct has previously been used to block bmp signaling in duck and chicken embryos ( 18 ) . briefly , embryos were cleared in dent 's bleach and incubated overnight with antibodies diluted in blocking buffer . confocal images were obtained from embryos cleared in benzyl alcohol / benzyl benzoate ( babb ) ( 19 ) on a zeiss lsm510 axioimager . for antibodies , these data can be found in online appendix supplementary table 1 , available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1010/dc1 . perfusion of embryos with lens culinaris agglutinin was performed with a protocol modified from jilani et al . the embryos were dissected in pbs , and 10 l was injected into the heart with a pulled glass pipette . labeled areas were quantified using imagej software by analyzing every second optical section corresponding to an optical depth of 20 m through the whole pancreas ( between 7 and 12 sections from each embryos ) . lentiviral constructs were generated by gateway recombination cloning , using entry vectors with the same constructs used in the chicken experiments . ( 21 ) , except that the epithelium was not separated from the mesenchyme before lentiviral infection . all lentiviral vectors were used at viral concentrations corresponding to 300 ng of p24 capsid protein per explant . the e12.5-pancreas explants were fixed in formalin and embedded in paraffin 7 days after transduction . the explants were sectioned ( 4 m ) and stained for insulin , amylase , and dna ( dapi ) as described by haumaitre et al . quantification was performed every fifth section spanning the entire explant using imagej software , and total area , insulin area , and amylase area surface were measured . in the e10.5 gut tube explants 10-m cryosections of the entire explants were stained for insulin , amylase , and dna ( dapi ) . quantification was performed every second section by counting the nuclei of insulin and amylase - positive cells . bmp signaling is transduced by smad1 , 5 , and 8 which , upon signaling , become phosphorylated and accumulates in the nucleus where they act as transcription factors ( 23 ) . we used a phosphosmad1,5,8-specific antibody ( 24 ) to characterize endogenous bmp signaling in the embryonic chicken pancreas ( fig . 1 ) at hamburger and hamilton ( hh ) stages 12 , 18 , and 21 ( 25 ) . this corresponds to time points when the pancreas has just been specified in the endoderm , dorsal and ventral buds have formed , and -cells , -cells , and amylase - positive cells can be detected ( 6,17 ) . psmad1,5,8 stainings on e2 , e3 , and e4 ( hh st.12 , 18 , and 21 ) chicken embryos ( a j ) , e9.5 mouse embryo ( k l ) , and e11.5-dissected mouse gut ( m n ) . images are either projections of confocal z - stacks of thick vibratome sections ( a c ) or an optical section ( d ) . boxed area in b is shown in higher magnification in c and d. a : projected image from a z - stack showing psmad1,5,8 reactivity in the ventral forgut endoderm ( arrows ) and dorsal neural tube ( arrowhead ) . b : projected image at the presumptive pancreas level . note psmad1,5,8 reactivity in the lateral plate mesoderm and the underlying endoderm that appears yellow because of coexpression with foxa2 ( arrow ) . c and d : psmad1,5,8 reactivity can be observed in the endothelial cells of the dorsal aortas and in endoderm lateral to the nkx6.1 expression domain ( arrowhead ) . e and f : a section of an e3 pancreas stained as indicated ( shown with and without nkx6.1 ) . note the psmad1,5,8 reactivity in the mesenchyme and the absence of staining in the pancreas epithelium ( arrows point to the dorsal pancreas ) . g : composite of two images showing the dorsal and ventral pancreas and the liver ; psmad1,5,8 can be detected in the pancreas mesenchyme and in mesenchymal and epithelial compartments of the liver . h j : another section from the same pancreas showing absence of psmad1,5,8 immunoreactivity in the nkx6.1-positive pancreatic epithelium and -cells ( recognizable by their morphology , arrowhead ) . k and l : optical sections from a z - stack of an e9.5 mouse stained in whole mount ; psmad1,5,8 can be detected in the dorsal aorta and intersomitic vessels and in mesenchymal cells in close contact with the ventral pancreas epithelium . a , aorta ; dp , dorsal pancreas ; duo , duodenum , v , right omphalomesenteric vein ; vp , ventral pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) at hh st . 12 , embryos were whole - mount stained and 200-m transverse vibratome sections were analyzed ( fig . d ) and in the ventral part of the anterior foregut endoderm ( arrows , fig . 1a , ) as previously reported ( 24 ) . at the level of the 3rd to 8th somite pairs , where the prospective pancreatic endoderm is located ( 3,26 ) , psmad1,5,8 was detected in the lateral plate mesoderm and in the endoderm lateral to the nkx6.1 expression domain ( fig . 1 ) . we could also observe psmad1,5,8 in the endothelium of the paired dorsal aortas ( fig . 18 , psmad1,5,8 immunoreactivity was detected in the pancreas mesenchyme and at lower levels in the duodenal epithelium ( fig . one day later , we still observed a mesenchymal psmad1,5,8 signal ( fig . 1g j ) , but no signal was detected in the dorsal or ventral pancreatic epithelium . the liver epithelium had a strong signal with a sharp border of immunoreactivity in the bile duct ( fig . 1 g arrows ) . no psmad1,5,8 reactivity was detected in nkx6.1-positive cells , which at this stage comprise both undifferentiated epithelial cells and -cells ( 17 ) ( fig . the absence of labeling in -cells was confirmed by colabeling with an insulin antibody ( supplementary fig . 1 g and h ) . to evaluate the status of smad1,5,8 phosphorylation in the early mouse pancreas , we performed whole - mount staining at e9.5 and e11.5 for psmad1,5,8 combined with pdx1 ( fig . no psmad1,5,8 was detected in the pdx1-positive endoderm . at e11.5 , we observed psmad1,5,8 reactivity in the pancreas mesenchyme , but no reactivity in the pancreas epithelium ( fig we conclude that endogenous signaling leading to detectable levels of psmad1,5,8 in the early chicken and mouse pancreas is entirely restricted to the mesenchyme . bmp4 , -5 , and -7 mrna have been reported to be expressed in the embryonic mouse pancreas by rt - pcr ( 15 ) , and bmp4 and bmp7 have been localized to the pancreas epithelium ( 16 ) , but little is known about bmp expression during chicken pancreas development . 22 ) to characterize the expression patterns of bmp2 , bmp4 , bmp5 , and bmp7 ( fig . 2 and data not shown ) . we found that bmp4 and bmp7 are expressed in the chicken pancreas mesenchyme and excluded from the epithelium at the stages examined ( fig . we could not detect bmp2 or bmp5 in the pancreas at any stage ( data not shown ) . from these results we concluded that bmp signaling is active in the pancreas mesenchyme , possibly mediated by bmps expressed within the mesenchyme . mrna ish , showing the expression of bmp4 and bmp7 at the level of the posterior stomach ( in a and c , arrows point at the dorsal aorta . arrowheads in a point to mesonephros , and arrowheads in c point to expression in the stomach mesenchyme ) and the pancreas ( b and d ) at hh . note that the mesenchymal expression of bmp4 and bmp7 in the pancreas mesenchyme ( arrows , b and d ) . ( a high - quality digital representation of this figure is available in the online issue . ) to investigate the requirements for bmp signaling in pancreas development , we applied two complementary approaches . we ectopically expressed the secreted bmp antagonist noggin or a dominant negative bmp receptor type 1 ( dnalk3 ) in the pancreas epithelium using in ovo electroporation of the endoderm ( 6,27 ) . we expected that the secreted antagonist noggin should block bmp signaling in the epithelium as well as in the mesenchyme , whereas dnalk3 should be autonomous to the epithelial cells expressing this construct . 1012 , and the introduced genes were thus expressed soon after the specification of the pancreas endoderm ( 17 ) . first we electroporated with a cmyc tagged noggin in the pcig5 vector which has an ires nuclear enhanced green fluorescent protein ( egfp ) sequence allowing direct monitoring of electroporated cells ( 6 ) . as expected , egfp was restricted to the epithelium after 32 h. however , cmyc immunoreactivity demonstrated that noggin was efficiently secreted and spread throughout the surrounding mesenchyme ( supplementary fig . we next assayed for psmad1,5,8 in the pancreas 24 and 48 h after electroporation ( fig . 3 ) . electroporation with a control plasmid did not change the pattern of endogenous psmad1,5,8 ( fig . 3b and f ) , but ectopic expression of noggin resulted in loss of mesenchymal psmad1,5,8 immunoreactivity around the electroporated area ( fig . we could still detect psmad1,5,8 in the mesenchyme at a distance from the electroporated area ( fig . we noted that the nkx6.1-positive pancreas epithelium appeared less developed than the corresponding controls after 48 h ( compare fig . ectopic expression of noggin in the chicken pancreas epithelium leads to loss of psmad1,5,8 immunoreactivity in the mesenchyme . as indicated , 24 h ( a d ) and 48 h ( e h ) after electroporation . note the loss of psmad1,5,8 in the pancreas mesenchyme around the noggin transfected area compared with controls . h : arrowheads point to psmad1,5,8 signals maintained at a distance from the electroporated area . ( a high - quality digital representation of this figure is available in the online issue . ) to assess pancreas morphogenesis , we performed whole - mount immunofluorescent staining of electroporated embryos at 24 , 48 , and 72 h. three - dimensional projections of confocal z - stacks were generated showing green fluorescent protein ( gfp ) , nkx6.1 , and glucagon / insulin ( fig . after electroporation with control plasmid or noggin , the dorsal and ventral pancreata were both formed normally , and endocrine cells were found exclusively in the dorsal pancreas ( fig . d ) . at 48 h , the pancreata electroporated with empty vector developed normally with primary branching , two distinct ventral pancreas buds ( 5 ) , and endocrine cells confined to the dorsal pancreas ( fig . instead the dorsal pancreas appeared as a tubular structure extending anteriorly along the stomach ( fig . 4 g and h ) . quantifying the amount of epithelial and endocrine areas showed that the total endocrine area was unchanged , but precocious endocrine cells could be observed in the ventral pancreas which was hypoplastic ( fig . 4 g and h , arrow ; supplementary table 2 ) . in some embryos , ectopic noggin expression in the pancreas results in poor epithelial branching , precocious endocrine differentiation . d : at 24 h after electroporation with control plasmid ( a and b ) and noggin ( c and d ) . projected images from z - stacks show correct specification of the dorsal and ventral pancreas under both conditions . b and d are identical to a and c , except that the gfp signal was removed to show the distribution of pancreatic cell types . e and f : forty - eight hours after electroporation with control plasmid ( e and f ) and noggin ( g and h ) . e and g : projected images from a z - stack . f and h : optical sections from the stack . in the control , primary branchpoint in the epithelium can be observed ( f , arrowheads ) and endocrine cells are restricted to the dorsal pancreas . the dorsal pancreas is completely unbranched after noggin electroporation and extends as a tubular structure anteriorly along the stomach ( arrowheads in g and h ) . numerous endocrine cells can be seen in the ventral pancreas ( arrow in g and h ) . l : seventy - two hours after electroporation with control plasmid ( i and j ) and noggin ( k and l ) . i and k : projected images from a z - stack . j and l : optical sections from the stack . branching is more pronounced in the controls after 72 h ( j , arrowheads ) , and endocrine cells a large cluster of pancreatic endocrine cells can be found detached from the pancreas in the stomach mesenchyme ( k and l , arrowhead ) . in the small remaining ventral pancreas , m : less frequently observed phenotype 48 h after noggin electroporation the dorsal pancreas is almost entirely converted into a large endocrine cell mass . n : electroporation with dnalk3 has no effect on the normal pancreas development 48 h after electroporation . see table 1 for quantifications . ( a high - quality digital representation of this figure is available in the online issue . ) at 72 h after electroporation with control vector , endocrine cells were still confined to the dorsal pancreas and branching of the epithelium was prominent ( fig . 4i and j ) . conversely , the pancreata of noggin electroporated embryos were hypomorphic , and large endocrine clusters detached from the pancreas could be observed in the stomach mesenchyme ( fig . there was no epithelial branching and endocrine cells were found in the ventral pancreas ( fig . cell - autonomous inhibition of bmp signaling in the endoderm via electroporation with dnalk3 had no effect on pancreas development ( fig . 4n ) , suggesting that the effect elicited by noggin is through inhibition of bmp signaling in the mesenchyme . in neural tube electroporation experiments , dnalk3 , as well as noggin , was able to block smad1,5,8 phosphorylation in the dorsal neural tube . confirming that the dnalk3 construct worked as expected ( supplementary fig . 2c h ) the pancreas of noggin electroporated embryos was further analyzed on sections 48 h after electroporation ( fig . the anterior extension of the pancreas contained nkx6.1 , glucagon , and insulin - positive cells ( fig . 5c ) , but no exocrine cells were found in noggin electroporated embryos ( fig . pancreatic cell types can be observed in the stomach mesenchyme and exocrine differentiation is reduced 48 h after electroporation with noggin . a and b are adjacent sections at the level of the stomach in a noggin electroporated embryo showing nkx6.1 mrna expression and insulin and glucagon protein expression , respectively . large clusters of pancreatic cells can be found in the stomach mesenchyme , and both glucagon and insulin are readily found ( a and b ) . c and d : sections of the pancreas of control ( c ) and noggin ( d ) electroprated embryos imunoflurescently labeled for amylase and showing gfp expression in electroporated cells . in d inset : the boxed area is without gfp . note the absence of amylase - positive cells in the noggin - electroporated pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) the results presented so far indicate that bmp signaling is required in the mesenchyme and that the effect on the epithelium is indirect . this was difficult to demonstrate directly since electroporation in the chicken embryo does not permit cell - autonomous inhibition of bmp signaling in the mesenchyme . therefore , we took advantage of established protocols for in vitro development of mouse pancreas explants and lentiviral gene transduction ( 28 ) . we explanted e12.5 pancreata transduced with lentivirial vectors encoding either gfp , noggin , or dnalk3 . when whole pancreas explants were exposed to lentiviral vectors , exclusive infection of the mesenchyme cell autonomous bmp signaling inhibition in the mouse pancreas mesenchyme results in increased -cell differentiation . mouse pancreas organ cultures explanted and transduced at e12.5 ( a k ) or 10.5 ( l n ) with lentiviral constructs as indicated . a d f : stereomicrographs of three different explants of each condition after 7 days in culture . g and h : explants transduced with a control egfp construct , immunflurescently stained for e - cadherin ( red ) and showing egfp with or without dapi nuclear counterstain . note how gfp expression is restricted to the mesenchyme 3 and 7 days after transduction . immunostained for amylase ( green ) , insulin ( red ) , and nuclei ( dapi , blue ) . n : explants transduced at e10.5 and cultured for 8 days , then stained for amylase and insulin with dapi nuclear counter stain . see table 2 for quantifications . ( a high - quality digital representation of this figure is available in the online issue . ) stereomicrographs of the explants cultured for 7 days showed that dnalk3 and noggin - transduced explants had a similar morphology and were of similar size as controls ( fig . f ) . costaining for e - cadherin and gfp on gfp - transduced explants confirmed the mesenchymal - specific expression after 3 and 7 days in culture ( fig . we quantified the number of exocrine cells and -cells after 7 days in culture ( fig . l , supplementary table 3 ) and found a 2.8-fold increase in the relative area of -cells in dnalk3-transduced explants compared with controls . this expansion was even more pronounced in noggin - transduced explants which showed a 3.6-fold increase in relative -cell area . in contrast , the relative exocrine area was diminished in dnalk3 and noggin - transduced explants compared with controls ( not statistically significant ) . there was no obvious branching defect in these studies , which could be a result of different timing of bmp inhibition compared with the studies performed in chicken . e10.5 gut tubes were dissected ; transduced with gfp , noggin , or dnalk3 ; and cultured for 4 and 8 days . after 4 days , the total explant volume was reduced by 5.5- and 1.8-fold when transduced with noggin and dnalk3 , respectively ( compared with control gfp transduction ; supplementary fig . noggin transduction resulted in a subtle change in the overall branching pattern , visualized by whole - mount mucin-1 staining , as main ducts were not well defined and appeared diffuse compared with controls ( supplementary fig . mesenchymal noggin expression increased the total number of insulin - positive cells by 3.8-fold , whereas the total number of amylase - positive cells were reduced by 1.6-fold ( fig . similarly , mesenchymal expression of dnalk3 increased the number of insulin - positive cells by 2.5-fold , demonstrating that cell autonomous bmp signal reception in the mesenchyme is required for a normal rate of endocrine cell differentiation to occur . we did observe a small but statistically significant effect on total exocrine cell numbers in noggin - transduced explants and in relative numbers of exocrine cells in both noggin and dnalk3 transduced ( supplementary table 3 ) . the effect of mesenchymal expression of dnalk3 on branching morphogenesis was ambiguous ; some explants exhibited a phenotype similar to the noggin - transduced explants , whereas some had a normal appearance ( not shown ) . since our results suggested that noggin inhibited bmp signaling in the mesenchyme , we next investigated mesenchymal gene expression patterns . pitx2 is normally expressed in the left side of the mesenchyme of vertebrate embryos and is involved in asymmetrical organ development ( 29,30 ) . bapx1 was recently demonstrated to be involved in the left - sided lateral growth of the e10.5 mouse dorsal pancreas because of a requirement for bapx1 in the formation of a specialized mesothelial structure , the splancnic mesodermal plate ( smp ) ( 31 ) . it is normally expressed in the left side of the mesenchyme , and it is required for the separation of the splenic and pancreatic mesenchyme and maintenance of the pancreas character in the distal part of the dorsal pancreas ( 31,32 ) . to evaluate the expression of pitx2 and bapx1 in relation to the chicken pancreas , we first performed mrna in situ hybridizations on serial sections for nkx6.1 and shh , together with pitx2 and bapx1 ( supplementary fig . 4 ) . we found that , as in the mouse , bapx1 was initially expressed bilaterally in the pancreas region ( supplementary fig . 4p ) , but from e3 strong expression was restricted to the pitx2-positive mesenchyme on the left side of the pancreas ( supplementary fig . bapx1 appeared to be symmetrically expressed at the level of the stomach ( supplementary fig . , we found that bapx1 was expressed left sided in the pancreas mesenchyme together with pitx2 after 48 h ( fig . 7c and e ) . in contrast , noggin - electroporated embryos displayed a duplication of bapx1 expression , whereas pitx2 remained left sided , suggesting that bapx1 and pitx2 expression is uncoupled at least under these conditions ( fig . these results are in agreement with a role for bmp signaling in development of the pancreas mesenchyme . since bapx1 is involved in the formation of the spm in the mouse embryo , we hypothesized that this structure could be affected in the bmp - inhibited embryos . we therefore stained for isl1 and laminin or nkx6.1/nkx2.2 with dapi nuclear counter stain to visualize this structure ( supplementary fig . , the spm was asymmetric with a condensation of the mesothelium on the left side at hh st . 5c and d ) , suggesting that downregulation of right - sided bapx1 expression is required for establishment of the asymmetric spm . electroporation with noggin results in persistence of a bapx1-positive pancreas mesenchyme on the right side of the pancreas 48 h after electroporation . a , c , e and b , d , f are serial sections from the same embryos , g j are serial sections from other embryos . pitx2 and bapx1 are normally coexpressed on the left side of the pancreas in control embryos ( arrows , c and e ) . however , in noggin - electroporated embryos , bapx1 expression becomes symmetrically expressed on both sides of the pancreas ( arrows , f ) , whereas pitx2 remains left sided ( arrow , d ) . most of the unbranced dorsal pancreas in noggin electroporated embryos does not express hes1 ( g and h ) , but the duodenum maintains hes1 expression . the observation of precocious endocrine differentiation in noggin - electroporated embryos prompted us to analyze the expression of hes1 . we found that the unbranched tubular dorsal pancreas was hes1-negative 48 h after electroporation , suggesting that the excessive endocrine differentiation is coincident with downregulated hes1 expression ( fig . 7e and k ) . we therefore looked after 32 h of development and earlier ( supplementary fig . 6 and data not shown ) . most of the nkx6.1-positive dorsal pancreas epithelium had differentiated after 32 h and was hes1-negative , but the remaining nkx6.1-positive cells expressed normal levels of hes1 . at earlier time points when the pancreas was more normal , we could not detect a general downregulation of hes1 in the epithelium ( not shown ) . we can therefore not distinguish between loss of hes1 followed by differentiation or vice versa , likely because our ish protocol does not allow quantitative measurements of mrna expression levels . the bilateral expression of bapx1 in the mesenchyme of noggin electroporated embryos was notable . in chicken and mice , the right omphalomesenteric vein is normally found at this position , and the developing pancreas grows around this vessel ( 4,5 ) . gene expression changes in this structure might indicate that the normal intimately joined development of the pancreas and the vein was disturbed . to visualize the vasculature and the pancreas , we injected embryos with fluorescently - labeled lens culinaris agglutin , a lectin with high affinity to chicken endothelial cells ( 20 ) , and subsequently we stained for nkx6.1 by whole - mount immunohistochemistry ( fig . 8) . embryos perfused with a fluorescently - tagged lens culinaris agglutinin ( labeling the vascular endothelium ) and subjected to whole - mount immunostaing for nkx6.1 and gfp . a , b and e , f show projected z - stacks from lateral views at two different magnifications . c and g : projected images from transverse thick vibratome sections from the same embryos ( indicated by dashed lines in a and e ) . note how the dorsal aorta is well separated from the pancreas in the control and how the dorsal pancreas curls around the omphalomesenteric vein . in noggin - electroporated embryos , the omphalomesenteric vein is consistently found ventrally to the pancreas , and in some of the embryos , the paired dorsal aortas fail to fuse . the bisymmetrical mesenchyme is indicated by dashed lines in f. ( the contrast was temporarily enhanced in the image to allow the drawing based on a background signal . the mesenchyme is not readily observable at the published contrast setting ) . d and h : schematic drawing summarizing the relationship between the gut and pancreas ( green ) and the omphalomesenteric vein ( red ) . a , aorta ; dp , dorsal pancreas ; duo , duodenum ; dv , ductus venosus ; li , liver ; ov , omphalomesenteric vein ; vp , ventral pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) at 3 days of development ( hh st . 19 ) , the paired omphalomesenteric veins run on each side of the gut tube and join anteriorly at the level of the liver in the ductus venosus ( 33 ) ( supplementary fig . the bilateral , ventral nkx6.1 domains are in close contact to these veins and the dorsal pancreas has grown asymmetrically over the right omphalomesenteric vein ( supplementary fig . the dorsal aorta is well separated from the pancreas by the pancreatic mesenchyme . during the next day , a series of anastomoses of the veins occur and most of the left omphalomesenteric vein regresses , leaving behind the right omphalomesenteric vein which curls around the gut at the level of the pancreas ( 5,33 ) ( supplementary fig . one of the ventral pancreata lie in close contact to the right omphalomesenteric vein ( supplementary fig . we analyzed the vascular development in control and noggin electroporated embryos 48 h after electroporation . 8c ) , and , as expected , the dorsal aorta was well separated from the dorsal pancreas ( fig . the omphalomesenteric vein was found on the ventral side of the pancreas and gut ( n = 6 ) and , in some embryos , the dorsal aortas failed to fuse and move dorsally ( 2 of 6 embryos ) . to assess the development of the microvasculature , we made flk1 ish on sectioned embryos . this revealed a normal microvasculature around the dorsal pancreas 32 h after electroporation when the omphalomesenteric vein is already displaced to the ventral side in bmp - inhibited embryos ( supplementary fig . bmp signaling is transduced by smad1 , 5 , and 8 which , upon signaling , become phosphorylated and accumulates in the nucleus where they act as transcription factors ( 23 ) . we used a phosphosmad1,5,8-specific antibody ( 24 ) to characterize endogenous bmp signaling in the embryonic chicken pancreas ( fig . 1 ) at hamburger and hamilton ( hh ) stages 12 , 18 , and 21 ( 25 ) . this corresponds to time points when the pancreas has just been specified in the endoderm , dorsal and ventral buds have formed , and -cells , -cells , and amylase - positive cells can be detected ( 6,17 ) . psmad1,5,8 stainings on e2 , e3 , and e4 ( hh st.12 , 18 , and 21 ) chicken embryos ( a j ) , e9.5 mouse embryo ( k l ) , and e11.5-dissected mouse gut ( m n ) . images are either projections of confocal z - stacks of thick vibratome sections ( a c ) or an optical section ( d ) . boxed area in b is shown in higher magnification in c and d. a : projected image from a z - stack showing psmad1,5,8 reactivity in the ventral forgut endoderm ( arrows ) and dorsal neural tube ( arrowhead ) . b : projected image at the presumptive pancreas level . note psmad1,5,8 reactivity in the lateral plate mesoderm and the underlying endoderm that appears yellow because of coexpression with foxa2 ( arrow ) . c and d : psmad1,5,8 reactivity can be observed in the endothelial cells of the dorsal aortas and in endoderm lateral to the nkx6.1 expression domain ( arrowhead ) . e and f : a section of an e3 pancreas stained as indicated ( shown with and without nkx6.1 ) . note the psmad1,5,8 reactivity in the mesenchyme and the absence of staining in the pancreas epithelium ( arrows point to the dorsal pancreas ) . g : composite of two images showing the dorsal and ventral pancreas and the liver ; psmad1,5,8 can be detected in the pancreas mesenchyme and in mesenchymal and epithelial compartments of the liver . h j : another section from the same pancreas showing absence of psmad1,5,8 immunoreactivity in the nkx6.1-positive pancreatic epithelium and -cells ( recognizable by their morphology , arrowhead ) . k and l : optical sections from a z - stack of an e9.5 mouse stained in whole mount ; psmad1,5,8 can be detected in the dorsal aorta and intersomitic vessels and in mesenchymal cells in close contact with the ventral pancreas epithelium . a , aorta ; dp , dorsal pancreas ; duo , duodenum , v , right omphalomesenteric vein ; vp , ventral pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) at hh st . 12 , embryos were whole - mount stained and 200-m transverse vibratome sections were analyzed ( fig . d ) and in the ventral part of the anterior foregut endoderm ( arrows , fig . 1a , ) as previously reported ( 24 ) . at the level of the 3rd to 8th somite pairs , where the prospective pancreatic endoderm is located ( 3,26 ) , psmad1,5,8 was detected in the lateral plate mesoderm and in the endoderm lateral to the nkx6.1 expression domain ( fig . 1 ) . we could also observe psmad1,5,8 in the endothelium of the paired dorsal aortas ( fig . 18 , psmad1,5,8 immunoreactivity was detected in the pancreas mesenchyme and at lower levels in the duodenal epithelium ( fig . one day later , we still observed a mesenchymal psmad1,5,8 signal ( fig . 1g j ) , but no signal was detected in the dorsal or ventral pancreatic epithelium . the liver epithelium had a strong signal with a sharp border of immunoreactivity in the bile duct ( fig . 1 g arrows ) . no psmad1,5,8 reactivity was detected in nkx6.1-positive cells , which at this stage comprise both undifferentiated epithelial cells and -cells ( 17 ) ( fig . the absence of labeling in -cells was confirmed by colabeling with an insulin antibody ( supplementary fig . 1 g and h ) . to evaluate the status of smad1,5,8 phosphorylation in the early mouse pancreas , we performed whole - mount staining at e9.5 and e11.5 for psmad1,5,8 combined with pdx1 ( fig . no psmad1,5,8 was detected in the pdx1-positive endoderm . at e11.5 , we observed psmad1,5,8 reactivity in the pancreas mesenchyme , but no reactivity in the pancreas epithelium ( fig we conclude that endogenous signaling leading to detectable levels of psmad1,5,8 in the early chicken and mouse pancreas is entirely restricted to the mesenchyme . bmp4 , -5 , and -7 mrna have been reported to be expressed in the embryonic mouse pancreas by rt - pcr ( 15 ) , and bmp4 and bmp7 have been localized to the pancreas epithelium ( 16 ) , but little is known about bmp expression during chicken pancreas development . 22 ) to characterize the expression patterns of bmp2 , bmp4 , bmp5 , and bmp7 ( fig . 2 and data not shown ) . we found that bmp4 and bmp7 are expressed in the chicken pancreas mesenchyme and excluded from the epithelium at the stages examined ( fig . we could not detect bmp2 or bmp5 in the pancreas at any stage ( data not shown ) . from these results we concluded that bmp signaling is active in the pancreas mesenchyme , possibly mediated by bmps expressed within the mesenchyme . mrna ish , showing the expression of bmp4 and bmp7 at the level of the posterior stomach ( in a and c , arrows point at the dorsal aorta . arrowheads in a point to mesonephros , and arrowheads in c point to expression in the stomach mesenchyme ) and the pancreas ( b and d ) at hh . note that the mesenchymal expression of bmp4 and bmp7 in the pancreas mesenchyme ( arrows , b and d ) . ( a high - quality digital representation of this figure is available in the online issue . ) to investigate the requirements for bmp signaling in pancreas development , we applied two complementary approaches . we ectopically expressed the secreted bmp antagonist noggin or a dominant negative bmp receptor type 1 ( dnalk3 ) in the pancreas epithelium using in ovo electroporation of the endoderm ( 6,27 ) . we expected that the secreted antagonist noggin should block bmp signaling in the epithelium as well as in the mesenchyme , whereas dnalk3 should be autonomous to the epithelial cells expressing this construct . 1012 , and the introduced genes were thus expressed soon after the specification of the pancreas endoderm ( 17 ) . first we electroporated with a cmyc tagged noggin in the pcig5 vector which has an ires nuclear enhanced green fluorescent protein ( egfp ) sequence allowing direct monitoring of electroporated cells ( 6 ) . as expected , egfp was restricted to the epithelium after 32 h. however , cmyc immunoreactivity demonstrated that noggin was efficiently secreted and spread throughout the surrounding mesenchyme ( supplementary fig . we next assayed for psmad1,5,8 in the pancreas 24 and 48 h after electroporation ( fig . 3 ) . electroporation with a control plasmid did not change the pattern of endogenous psmad1,5,8 ( fig . 3b and f ) , but ectopic expression of noggin resulted in loss of mesenchymal psmad1,5,8 immunoreactivity around the electroporated area ( fig . we could still detect psmad1,5,8 in the mesenchyme at a distance from the electroporated area ( fig . we noted that the nkx6.1-positive pancreas epithelium appeared less developed than the corresponding controls after 48 h ( compare fig . 3g ectopic expression of noggin in the chicken pancreas epithelium leads to loss of psmad1,5,8 immunoreactivity in the mesenchyme . sectioned pancreata immunolabeled as indicated , 24 h ( a d ) and 48 h ( e h ) after electroporation . note the loss of psmad1,5,8 in the pancreas mesenchyme around the noggin transfected area compared with controls . h : arrowheads point to psmad1,5,8 signals maintained at a distance from the electroporated area . ( a high - quality digital representation of this figure is available in the online issue . ) to assess pancreas morphogenesis , we performed whole - mount immunofluorescent staining of electroporated embryos at 24 , 48 , and 72 h. three - dimensional projections of confocal z - stacks were generated showing green fluorescent protein ( gfp ) , nkx6.1 , and glucagon / insulin ( fig . after electroporation with control plasmid or noggin , the dorsal and ventral pancreata were both formed normally , and endocrine cells were found exclusively in the dorsal pancreas ( fig . d ) . at 48 h , the pancreata electroporated with empty vector developed normally with primary branching , two distinct ventral pancreas buds ( 5 ) , and endocrine cells confined to the dorsal pancreas ( fig . 4e and f ) . instead the dorsal pancreas appeared as a tubular structure extending anteriorly along the stomach ( fig . 4 g and h ) . quantifying the amount of epithelial and endocrine areas showed that the total endocrine area was unchanged , but precocious endocrine cells could be observed in the ventral pancreas which was hypoplastic ( fig . 4 g and h , arrow ; supplementary table 2 ) . in some embryos , ectopic noggin expression in the pancreas results in poor epithelial branching , precocious endocrine differentiation . d : at 24 h after electroporation with control plasmid ( a and b ) and noggin ( c and d ) . projected images from z - stacks show correct specification of the dorsal and ventral pancreas under both conditions . b and d are identical to a and c , except that the gfp signal was removed to show the distribution of pancreatic cell types . e and f : forty - eight hours after electroporation with control plasmid ( e and f ) and noggin ( g and h ) . e and g : projected images from a z - stack . f and h : optical sections from the stack . in the control , primary branchpoint in the epithelium can be observed ( f , arrowheads ) and endocrine cells are restricted to the dorsal pancreas . the dorsal pancreas is completely unbranched after noggin electroporation and extends as a tubular structure anteriorly along the stomach ( arrowheads in g and h ) . numerous endocrine cells can be seen in the ventral pancreas ( arrow in g and h ) . l : seventy - two hours after electroporation with control plasmid ( i and j ) and noggin ( k and l ) . i and k : projected images from a z - stack . j and l : optical sections from the stack . branching is more pronounced in the controls after 72 h ( j , arrowheads ) , and endocrine cells are still confined to the dorsal pancreas . in noggin a large cluster of pancreatic endocrine cells can be found detached from the pancreas in the stomach mesenchyme ( k and l , arrowhead ) . in the small remaining ventral pancreas , m : less frequently observed phenotype 48 h after noggin electroporation the dorsal pancreas is almost entirely converted into a large endocrine cell mass . n : electroporation with dnalk3 has no effect on the normal pancreas development 48 h after electroporation . see table 1 for quantifications . ( a high - quality digital representation of this figure is available in the online issue . ) at 72 h after electroporation with control vector , endocrine cells were still confined to the dorsal pancreas and branching of the epithelium was prominent ( fig . 4i and j ) . conversely , the pancreata of noggin electroporated embryos were hypomorphic , and large endocrine clusters detached from the pancreas could be observed in the stomach mesenchyme ( fig . there was no epithelial branching and endocrine cells were found in the ventral pancreas ( fig . cell - autonomous inhibition of bmp signaling in the endoderm via electroporation with dnalk3 had no effect on pancreas development ( fig . 4n ) , suggesting that the effect elicited by noggin is through inhibition of bmp signaling in the mesenchyme . in neural tube electroporation experiments , dnalk3 , as well as noggin , was able to block smad1,5,8 phosphorylation in the dorsal neural tube . confirming that the dnalk3 construct worked as expected ( supplementary fig . 2c h ) . the pancreas of noggin electroporated embryos was further analyzed on sections 48 h after electroporation ( fig . the anterior extension of the pancreas contained nkx6.1 , glucagon , and insulin - positive cells ( fig . 5c ) , but no exocrine cells were found in noggin electroporated embryos ( fig . pancreatic cell types can be observed in the stomach mesenchyme and exocrine differentiation is reduced 48 h after electroporation with noggin . a and b are adjacent sections at the level of the stomach in a noggin electroporated embryo showing nkx6.1 mrna expression and insulin and glucagon protein expression , respectively . large clusters of pancreatic cells can be found in the stomach mesenchyme , and both glucagon and insulin are readily found ( a and b ) . c and d : sections of the pancreas of control ( c ) and noggin ( d ) electroprated embryos imunoflurescently labeled for amylase and showing gfp expression in electroporated cells . in d inset : the boxed area is without gfp . note the absence of amylase - positive cells in the noggin - electroporated pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) the results presented so far indicate that bmp signaling is required in the mesenchyme and that the effect on the epithelium is indirect . this was difficult to demonstrate directly since electroporation in the chicken embryo does not permit cell - autonomous inhibition of bmp signaling in the mesenchyme . therefore , we took advantage of established protocols for in vitro development of mouse pancreas explants and lentiviral gene transduction ( 28 ) . we explanted e12.5 pancreata transduced with lentivirial vectors encoding either gfp , noggin , or dnalk3 . when whole pancreas explants were exposed to lentiviral vectors , exclusive infection of the mesenchyme cell autonomous bmp signaling inhibition in the mouse pancreas mesenchyme results in increased -cell differentiation . mouse pancreas organ cultures explanted and transduced at e12.5 ( a k ) or 10.5 ( l n ) with lentiviral constructs as indicated . a d f : stereomicrographs of three different explants of each condition after 7 days in culture . g and h : explants transduced with a control egfp construct , immunflurescently stained for e - cadherin ( red ) and showing egfp with or without dapi nuclear counterstain . note how gfp expression is restricted to the mesenchyme 3 and 7 days after transduction . immunostained for amylase ( green ) , insulin ( red ) , and nuclei ( dapi , blue ) . n : explants transduced at e10.5 and cultured for 8 days , then stained for amylase and insulin with dapi nuclear counter stain . see table 2 for quantifications . ( a high - quality digital representation of this figure is available in the online issue . ) stereomicrographs of the explants cultured for 7 days showed that dnalk3 and noggin - transduced explants had a similar morphology and were of similar size as controls ( fig . f ) . costaining for e - cadherin and gfp on gfp - transduced explants confirmed the mesenchymal - specific expression after 3 and 7 days in culture ( fig . we quantified the number of exocrine cells and -cells after 7 days in culture ( fig . l , supplementary table 3 ) and found a 2.8-fold increase in the relative area of -cells in dnalk3-transduced explants compared with controls . this expansion was even more pronounced in noggin - transduced explants which showed a 3.6-fold increase in relative -cell area . in contrast , the relative exocrine area was diminished in dnalk3 and noggin - transduced explants compared with controls ( not statistically significant ) . there was no obvious branching defect in these studies , which could be a result of different timing of bmp inhibition compared with the studies performed in chicken . e10.5 gut tubes were dissected ; transduced with gfp , noggin , or dnalk3 ; and cultured for 4 and 8 days . after 4 days , the total explant volume was reduced by 5.5- and 1.8-fold when transduced with noggin and dnalk3 , respectively ( compared with control gfp transduction ; supplementary fig . noggin transduction resulted in a subtle change in the overall branching pattern , visualized by whole - mount mucin-1 staining , as main ducts were not well defined and appeared diffuse compared with controls ( supplementary fig . after 8 days in culture , mesenchymal noggin expression increased the total number of insulin - positive cells by 3.8-fold , whereas the total number of amylase - positive cells were reduced by 1.6-fold ( fig . similarly , mesenchymal expression of dnalk3 increased the number of insulin - positive cells by 2.5-fold , demonstrating that cell autonomous bmp signal reception in the mesenchyme is required for a normal rate of endocrine cell differentiation to occur . we did observe a small but statistically significant effect on total exocrine cell numbers in noggin - transduced explants and in relative numbers of exocrine cells in both noggin and dnalk3 transduced ( supplementary table 3 ) . the effect of mesenchymal expression of dnalk3 on branching morphogenesis was ambiguous ; some explants exhibited a phenotype similar to the noggin - transduced explants , whereas some had a normal appearance ( not shown ) . since our results suggested that noggin inhibited bmp signaling in the mesenchyme , we next investigated mesenchymal gene expression patterns . pitx2 is normally expressed in the left side of the mesenchyme of vertebrate embryos and is involved in asymmetrical organ development ( 29,30 ) . bapx1 was recently demonstrated to be involved in the left - sided lateral growth of the e10.5 mouse dorsal pancreas because of a requirement for bapx1 in the formation of a specialized mesothelial structure , the splancnic mesodermal plate ( smp ) ( 31 ) . it is normally expressed in the left side of the mesenchyme , and it is required for the separation of the splenic and pancreatic mesenchyme and maintenance of the pancreas character in the distal part of the dorsal pancreas ( 31,32 ) . to evaluate the expression of pitx2 and bapx1 in relation to the chicken pancreas , we first performed mrna in situ hybridizations on serial sections for nkx6.1 and shh , together with pitx2 and bapx1 ( supplementary fig . 4 ) . we found that , as in the mouse , bapx1 was initially expressed bilaterally in the pancreas region ( supplementary fig . 4p ) , but from e3 strong expression was restricted to the pitx2-positive mesenchyme on the left side of the pancreas ( supplementary fig . bapx1 appeared to be symmetrically expressed at the level of the stomach ( supplementary fig . 4h ) as in the mouse ( 31 ) . in control embryos , we found that bapx1 was expressed left sided in the pancreas mesenchyme together with pitx2 after 48 h ( fig . 7c and e ) . in contrast , noggin - electroporated embryos displayed a duplication of bapx1 expression , whereas pitx2 remained left sided , suggesting that bapx1 and pitx2 expression is uncoupled at least under these conditions ( fig . these results are in agreement with a role for bmp signaling in development of the pancreas mesenchyme . since bapx1 is involved in the formation of the spm in the mouse embryo , we hypothesized that this structure could be affected in the bmp - inhibited embryos . we therefore stained for isl1 and laminin or nkx6.1/nkx2.2 with dapi nuclear counter stain to visualize this structure ( supplementary fig . , the spm was asymmetric with a condensation of the mesothelium on the left side at hh st . 5c and d ) , suggesting that downregulation of right - sided bapx1 expression is required for establishment of the asymmetric spm . electroporation with noggin results in persistence of a bapx1-positive pancreas mesenchyme on the right side of the pancreas 48 h after electroporation . mrna in situ hybridization on electroporated embryos 48 h after electroporation . a , c , e and b , d , f are serial sections from the same embryos , g j are serial sections from other embryos . pitx2 and bapx1 are normally coexpressed on the left side of the pancreas in control embryos ( arrows , c and e ) . however , in noggin - electroporated embryos , bapx1 expression becomes symmetrically expressed on both sides of the pancreas ( arrows , f ) , whereas pitx2 remains left sided ( arrow , d ) . most of the unbranced dorsal pancreas in noggin electroporated embryos does not express hes1 ( g and h ) , but the duodenum maintains hes1 expression . the observation of precocious endocrine differentiation in noggin - electroporated embryos prompted us to analyze the expression of hes1 . we found that the unbranched tubular dorsal pancreas was hes1-negative 48 h after electroporation , suggesting that the excessive endocrine differentiation is coincident with downregulated hes1 expression ( fig . 7e and k ) . most of the nkx6.1-positive dorsal pancreas epithelium had differentiated after 32 h and was hes1-negative , but the remaining nkx6.1-positive cells expressed normal levels of hes1 . at earlier time points when the pancreas was more normal , we could not detect a general downregulation of hes1 in the epithelium ( not shown ) . we can therefore not distinguish between loss of hes1 followed by differentiation or vice versa , likely because our ish protocol does not allow quantitative measurements of mrna expression levels . the bilateral expression of bapx1 in the mesenchyme of noggin electroporated embryos was notable . in chicken and mice , the right omphalomesenteric vein is normally found at this position , and the developing pancreas grows around this vessel ( 4,5 ) . gene expression changes in this structure might indicate that the normal intimately joined development of the pancreas and the vein was disturbed . to visualize the vasculature and the pancreas , we injected embryos with fluorescently - labeled lens culinaris agglutin , a lectin with high affinity to chicken endothelial cells ( 20 ) , and subsequently we stained for nkx6.1 by whole - mount immunohistochemistry ( fig . 8) . embryos perfused with a fluorescently - tagged lens culinaris agglutinin ( labeling the vascular endothelium ) and subjected to whole - mount immunostaing for nkx6.1 and gfp . a , b and e , f show projected z - stacks from lateral views at two different magnifications . c and g : projected images from transverse thick vibratome sections from the same embryos ( indicated by dashed lines in a and e ) . note how the dorsal aorta is well separated from the pancreas in the control and how the dorsal pancreas curls around the omphalomesenteric vein . in noggin - electroporated embryos , the omphalomesenteric vein is consistently found ventrally to the pancreas , and in some of the embryos , the paired dorsal aortas fail to fuse . the bisymmetrical mesenchyme is indicated by dashed lines in f. ( the contrast was temporarily enhanced in the image to allow the drawing based on a background signal . d and h : schematic drawing summarizing the relationship between the gut and pancreas ( green ) and the omphalomesenteric vein ( red ) . a , aorta ; dp , dorsal pancreas ; duo , duodenum ; dv , ductus venosus ; li , liver ; ov , omphalomesenteric vein ; vp , ventral pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) at 3 days of development ( hh st . 19 ) , the paired omphalomesenteric veins run on each side of the gut tube and join anteriorly at the level of the liver in the ductus venosus ( 33 ) ( supplementary fig . the bilateral , ventral nkx6.1 domains are in close contact to these veins and the dorsal pancreas has grown asymmetrically over the right omphalomesenteric vein ( supplementary fig . the dorsal aorta is well separated from the pancreas by the pancreatic mesenchyme . during the next day , a series of anastomoses of the veins occur and most of the left omphalomesenteric vein regresses , leaving behind the right omphalomesenteric vein which curls around the gut at the level of the pancreas ( 5,33 ) ( supplementary fig . one of the ventral pancreata lie in close contact to the right omphalomesenteric vein ( supplementary fig . we analyzed the vascular development in control and noggin electroporated embryos 48 h after electroporation . 8c ) , and , as expected , the dorsal aorta was well separated from the dorsal pancreas ( fig . in noggin electroporated embryos , the vasculature developed abnormally ( fig . 8e g ) . the omphalomesenteric vein was found on the ventral side of the pancreas and gut ( n = 6 ) and , in some embryos , the dorsal aortas failed to fuse and move dorsally ( 2 of 6 embryos ) . to assess the development of the microvasculature this revealed a normal microvasculature around the dorsal pancreas 32 h after electroporation when the omphalomesenteric vein is already displaced to the ventral side in bmp - inhibited embryos ( supplementary fig . development of the pancreas depends on interactions between the epithelium and the mesenchyme as recognized since the studies by golosow and grobstein ( 12 ) , but few endogenous signaling factors have been identified . here , we used chicken in ovo endoderm electroporation timed to express the pancreatic transgenes after specification of the pancreas . detection of psmad1,5,8 shows that early pancreatic mesenchyme receives a bmp signal , possibly through alk3 , which is expressed in the chicken pancreas mesenchyme ( 34 ) . since we find bmp expression in the mesenchyme , it is possible that an autocrine signaling mechanism is occurring . epithelial expression of the secreted bmp antagonist noggin resulted in the loss of psmad1,5,8 immunoreactivity in the adjacent mesenchyme and affected epithelial and mesenchymal development . the epithelium displayed reduced branching , anterior extension of the dorsal pancreas into the stomach mesenchyme , and precocious endocrine differentiation . inhibiting bmp signaling compromised angiogenic remodeling that positions the right omphalomesenteric vein adjacent to the pancreas . instead , we observed a persistence of mesenchymal bapx1 expression , a bilateral smp , and failure of the dorsal aortas to fuse in the most severely affected embryos . we can not distinguish a direct effect on the endothelial cells from an indirect effect acting through the mesenchyme . however , the microvasculature seems normal in bmp - inhibited embryos , suggesting that some aspects of angiogenesis occur normally and the defect appears limited to major vessels . notably , the mouse explants corroborate the findings in chick , arguing against involvement of the major vessels . additionally , mesenchymal isl1 expression , dependent on signals from blood vessels ( 35 ) , is maintained . the results presented herein appear to conflict with a recent report by goulley et al . bmp signaling was perturbed by the expression of noggin and bmp4 in the pancreas epithelium using the pdx1 promoter , with no profound effect on pancreatic development . we speculate that this could be due to different levels of expression since establishment of high - expressing transgenic mouse lines used in their studies would be prevented by a severe vascular or pancreatic phenotype caused by embryonic or perinatal lethality , and thus a selection for low expressers . a similar study expressing bmp6 under the control of the pdx1 promoter resulted in complete pancreas agenesis at a late gestational age , and these mice displayed a hypomorphic spleen , suggesting that the mesenchyme was also affected ( 15 ) . the present study suggests that there is no epithelial bmp signaling in the pancreas at the investigated stages , and that the effect on epithelial development is secondary to inhibition of bmp signaling in the mesenchyme . this is supported by the observation that cell autonomous bmp - signaling inhibition in the mouse pancreas mesenchyme leads to an increase in endocrine differentiation concomitant with a decrease in exocrine development . it is possible that the effect of bmp inhibition on the mesenchyme can be subdivided into an inhibition of an autocrine mesenchymal signal and an associated effect on vascular remodelling . bmp signaling in the mesenchyme may be required for the mesenchyme to express a secondary factor acting on the epithelium . fgf10 is one candidate because mesenchymal fgf10 is required for epithelial branching and progenitor cell proliferation ( 911 ) . however , there is little fgf10 expression left at e12.5 , the time of explanting in some of the organ culture experiments , suggesting that other factors could be involved .

summarychronic rejection of solid organ allografts remains the major cause of transplant failure . donor - derived tissue - resident lymphocytes are transferred to the recipient during transplantation , but their impact on alloimmunity is unknown . using mouse cardiac transplant models , we show that graft - versus - host recognition by passenger donor cd4 t cells markedly augments recipient cellular and humoral alloimmunity , resulting in more severe allograft vasculopathy and early graft failure . this augmentation is enhanced when donors were pre - sensitized to the recipient , is dependent upon avoidance of host nk cell recognition , and is partly due to provision of cognate help for allo - specific b cells from donor cd4 t cells recognizing b cell mhc class ii in a peptide - degenerate manner . passenger donor lymphocytes may therefore influence recipient alloimmune responses and represent a therapeutic target in solid organ transplantation . objectivepancreas organogenesis is orchestrated by interactions between the epithelium and the mesenchyme , but these interactions are not completely understood . here we investigated a role for bone morphogenetic protein ( bmp ) signaling within the pancreas mesenchyme and found it to be required for the normal development of the mesenchyme as well as for the pancreatic epithelium.research design and methodswe analyzed active bmp signaling by immunostaining for phospho - smad1,5,8 and tested whether pancreas development was affected by bmp inhibition after expression of noggin and dominant negative bmp receptors in chicken and mouse pancreas.resultsendogenous bmp signaling is confined to the mesenchyme in the early pancreas and inhibition of bmp signaling results in severe pancreatic hypoplasia with reduced epithelial branching . notably , we also observed an excessive endocrine differentiation when mesenchymal bmp signaling is blocked , presumably secondary to defective mesenchyme to epithelium signaling.conclusionswe conclude that bmp signaling plays a previously unsuspected role in the mesenchyme , required for normal development of the mesenchyme as well as for the epithelium .

the combination of erosion , attrition and abrasion is called tooth wear . erosion is the loss of hard tissues due to chemical effects , but not bacteria . attrition is the wear of tooth against tooth and abrasion is the wear of teeth from other surfaces . recently , the role of abfraction has raised interest into abrasion and the link with attrition but this area is under research.1 extensive tooth wear is considered a potential threat to functional dentition . the management of tooth wear , especially from attrition , is becoming a subject of increasing interest in the prosthodontic literature , both from a preventive [ how to stop the progress of tooth substance loss ( tsl ) ] and from a restorative point of view ( how to replace the lost tooth substance and to restore function).2 by definition , attritional wear is the loss of tooth tissue due to friction between opposing teeth and is thus related to dental occlusion . tsl considered to be normal in aging process , in which depositioning of secondary dentine , alveolar growth , muscle adaptation , and attrition are all parts of a compensation mechanism.2 clinicians are often faced with the challenge of restoring severely worn dentition . a critical aspect for successful treatment of these patients is to determine the occlusal vertical dimension and the interocclusal rest space . a systematic approach to managing this type of complete oral rehabilitation can lead to a predictable and favorable treatment prognosis.3 when tooth surface loss is severe , it can be associated with decreased vertical dimension of the occlusion resulting in a poor aesthetic appearance , loss of muscle tone and decreased masticatory efficiency.4 in addition , tooth tissue loss from bruxism has been demonstrated to be associated with various dental problems such as tooth sensitivity , excessive reduction of clinical crown height and possible changes of occlusal relationship . this case report will present a sequence of treatment , including conservative multidisciplinary approach to restoring esthetics and function in a patient with worn anterior dentition . this case report describes the management of excessive tooth tissue loss in a 45 year old woman with a history of bruxism . prior to treatment , a detailed dental , medical , and social history was obtained from the patient . she started to notice that her teeth were getting shorter about 20 years ago ; however , she chose to leave the problem unattended . clinically , the patient demonstrated partial edentulism , several maxillary and mandibular teeth showing severe attrition to the free gingival level , an uneven occlusal plane anteroposteriorly and mediolaterally . clinically , the patient s facial appearance showed signs of a collapsed occlusal vertical dimension ( figure 1 ) . the preoperative interocclusal rest space was 3 mm . periodontal condition and soft - tissue examination showed no pocket depth of over 2 mm or mobility of any remaining teeth . the patient s maxilla diagnosed with class ii div 1 and mandibula class iii div 1 according to the kennedy classification system for partial edentulism ( figure 1 ) . the treatment goals were to restore the lost occlusal vertical dimension ( ovd ) to correct the occlusal plane , to restore function , and to restore the esthetics of the patient s dentition . phase i included a splint therapy and a provisional fixed prosthesis to re - establish correct vertical dimension of occlusion and stable occlusal contacts . tmj and muscle examinations were done at the first week and at the first month . this procedure should only be considered when the remaining root is supported by a healthy periodontium and the post - surgery crown / root ratio will be favorable . in the clinical examination there was no sign of gingival inflammation or periodontal infection , or tooth mobility . in the periapical radiographic examination , root / crown ratio of the teeth were more than two , and there was no alveolar resorption . sulcular and internal bevel incisions were performed with preserving the gingival papillas , without any vertical incisions . because there was an adequate amount of attached gingiva , internal bevel incision was performed 34 mm far away from the gingival margin , considering the zenith points in the anterior maxilla . after the removal of remnant gingiva , full - thickness muco - periosteal flap elevated . flap was closed using nylon sutures with vertical matrix suture technique . and also frenulum attachment was removed by frenectomy operation . wound healing was completed at the 6 week after the surgery in the follow - up period ( figure 3 ) . the design of the tooth preparation should encompass necessary reduction relative to tooth position and the requirements of the restorative material . the abutment teeth ( # 11,#12 , # 13 , # 21 , # 22 , # 23 ) for zirconium restorations were prepared , which implied a 1.2-mm deep circumferential shoulder and a slightly rounded inner angle.5,6 the other abutment teeth ( # 14 , # 15 , # 24 , # 25 , # 27 , # 33 , # 34 , # 37 , # 43 , # 44 , # 45 , # 47 ) were prepared with a knife edge finish line ( figure 4 ) . impressions for the maxillary and mandibular interim prostheses were made using a vinyl polysiloxane impression ( elite hd , zhermak , germany ) material . the impression was cast with a type iv dental stone ( silky - rock ; whip mix corp , louisville , ky ) , and the stone cast was mounted in a semi adjustable articulator ( dentatus arh - type ; dentatus ab , stockholm , sweden ) with an opposing mandibular cast . feldspathic porcelain was chosen to restore other teeth in both jaws ; the patient also was given an occlusion guard to protect the restoration against future bruxism . the crowns were luted with self - polymerizing composite resin cement ( multilink , ivoclar vivadent , schaan , liechtenstein ) ( figure 5 ) . after prosthetic management , the patient was instructed about individual oral hygiene care of fixed prostheses . after insertion of the interim prostheses the patient reported no muscular or temporomandibular joint discomfort . follow - up treatments were done to evaluate the patient s comfort , arch form , and potential occlusal vertical dimension problems . patient was recalled twice for postoperative examinations during a 1-year follow - up period ( figure 6 ) . in the controls it was seen that marginal adaptation of the prostheses was appropriate and there were no signs of gingival inflammation and recession . the treatment goals were to restore the lost occlusal vertical dimension ( ovd ) to correct the occlusal plane , to restore function , and to restore the esthetics of the patient s dentition . phase i included a splint therapy and a provisional fixed prosthesis to re - establish correct vertical dimension of occlusion and stable occlusal contacts . tmj and muscle examinations were done at the first week and at the first month . this procedure should only be considered when the remaining root is supported by a healthy periodontium and the post - surgery crown / root ratio will be favorable . in the clinical examination there was no sign of gingival inflammation or periodontal infection , or tooth mobility . in the periapical radiographic examination , root / crown ratio of the teeth were more than two , and there was no alveolar resorption . sulcular and internal bevel incisions were performed with preserving the gingival papillas , without any vertical incisions . because there was an adequate amount of attached gingiva , internal bevel incision was performed 34 mm far away from the gingival margin , considering the zenith points in the anterior maxilla . after the removal of remnant gingiva , full - thickness muco - periosteal flap elevated . wound healing was completed at the 6 week after the surgery in the follow - up period ( figure 3 ) . the design of the tooth preparation should encompass necessary reduction relative to tooth position and the requirements of the restorative material . the abutment teeth ( # 11,#12 , # 13 , # 21 , # 22 , # 23 ) for zirconium restorations were prepared , which implied a 1.2-mm deep circumferential shoulder and a slightly rounded inner angle.5,6 the other abutment teeth ( # 14 , # 15 , # 24 , # 25 , # 27 , # 33 , # 34 , # 37 , # 43 , # 44 , # 45 , # 47 ) were prepared with a knife edge finish line ( figure 4 ) . impressions for the maxillary and mandibular interim prostheses were made using a vinyl polysiloxane impression ( elite hd , zhermak , germany ) material . the impression was cast with a type iv dental stone ( silky - rock ; whip mix corp , louisville , ky ) , and the stone cast was mounted in a semi adjustable articulator ( dentatus arh - type ; dentatus ab , stockholm , sweden ) with an opposing mandibular cast . feldspathic porcelain was chosen to restore other teeth in both jaws ; the patient also was given an occlusion guard to protect the restoration against future bruxism . the crowns were luted with self - polymerizing composite resin cement ( multilink , ivoclar vivadent , schaan , liechtenstein ) ( figure 5 ) . after prosthetic management after insertion of the interim prostheses the patient reported no muscular or temporomandibular joint discomfort . follow - up treatments were done to evaluate the patient s comfort , arch form , and potential occlusal vertical dimension problems . patient was recalled twice for postoperative examinations during a 1-year follow - up period ( figure 6 ) . in the controls it was seen that marginal adaptation of the prostheses was appropriate and there were no signs of gingival inflammation and recession . the etiology of occlusal wear for our patient is not fully understood ; however , it can be hypothesized that the patient had a parafunctional occlusal habit and started grinding her anterior teeth . once the anterior teeth got shorter , the patient lost anterior guidance and developed posterior interferences . the posterior interferences in lateral excursions can activate the masseter and temporalis muscles , enabling the patient to generate more forces to grind her teeth more aggressively.7 a mutually protected occlusal scheme was used to prevent the destruction of the new prostheses.3 mutually protected articulation is described as an occlusal scheme in which the posterior teeth prevent excessive contact of the anterior teeth in maximum intercuspation , and the anterior teeth disengage the posterior teeth in all mandibular excursive movements . studies have shown that in lateral excursive movements,8 the anterior teeth can best receive and dissipate the forces9 and posterior contacts in excursions appear to provide unfavorable forces to the masticatory system because of the amount and direction of the applied forces.10,11 in addition to the use of the mutually protected occlusal scheme , an occlusal splint was fabricated for night wear , and the patient was instructed and trained to keep her teeth apart when not actively chewing . zirconia ceramic crowns were selected for the patient s treatment to ensure adequate strength on upper anterior teeth . the primary advantage of a zirconia restoration is esthetic benefit , as it is translucent and tooth - colored.12 a metal - free ceramic crown can transmit a great amount of incident light through to a ceramic core where light is scattered in a natural fashion.12 thus , the appearance of definitive restorations may be very close to that of a natural tooth . strength , fit , and esthetics are traditionally considered in the selection of material for full coverage restorations . traditional cements occupy the space between the restoration and the tooth surfaces but do not provide adhesion between them . resin cements provide adhesion to both surfaces and can act to transfer force from the restoration to the underlying tooth and strengthen all - ceramic restorations.13 also resin cement was used to provide additional bonding strength to increase the retention of the restorations because our patient s clinical crown was not long enough . in the present case , the maxillary occlusal plane had already been established . the use of a provisional occlusal splint is generally considered the first step in the treatment of inappropriate horizontal and vertical maxillomandibular relationships . in the present case , approximately 4 mm of crown lengthening was obtained in the maxillary anterior teeth by resective bone surgery . some studies suggested that patients with healthy periodontium have 0.69 mm mean sulcus depth , 0.97 mm epithelial attachment and 1.07 mm connective tissue.14 therefore the total length of supracreasteal gingival tissue was 2.73 mm . so , reconstitution of 2.73 mm of gingival tissue must be considered , while deciding the amount of bone resection.15 protection sufficient amount of attached gingiva around the teeth after the surgery is also an important task to achieve.16 in our case , 5 mm of attached gingiva existed after the surgery . in order to prevent the frenulum become high after the surgery , frenectomy operation also performed at the same appointment . in the present case report , it has been shown that , after the use of occlusal guard and the surgical treatment ; restoration of maxillary teeth with full coverage restorations and mandibular teeth with metal ceramic restorations help to restore the occlusal vertical dimension . dentists have the responsibility of developing proper form and function to protect the integrity of the masticator system , and must connect the new materials and technology with traditional functional concepts to be successful . as the case presented here demonstrates , this combination of innovation and tradition is achievable with careful planning . the restored occlusal vertical dimension has shown to be physiologically and esthetically acceptable to the patient . one of the most frequently used measures of health status is the question how would you rate your health ? , a single question asking people to evaluate their overall health on a scale from excellent to poor . this single item measure has been demonstrated to be a robust predictor of health outcomes , including mortality , independent of health indicators of physical health . because of the wide range of associations with other health indicators , and the simplicity with which it is collected , self - rated health is widely used in large population surveys as a proxy measure for general health status . earlier studies assumed that the answer to this single question embodied a simple summation of all objective states , and therefore focused on the differences in prediction between the objective measures and the subjective self report . when the subjective self perception of health remained a strong predictor of mortality regardless of the statistical control of numerous objective health variables , it became clear that the simple question is not as simple as was originally thought . the attempts to understand what lies behind this measure produced studies that included mental / emotional variables in addition to physical health variables , and one explanation for the ability of the self rated health to predict mortality was that the single question of how would you rate your health is interpreted by subjects as referring to their overall health , including psychosocial aspects and not only to their physical health . two studies showed that mental health was the mediator in the relationship between physical health and self reported general health . in one case , mental health was measured by the anxiety and depression scale hads and the study was done on 449 adults undergoing hip or knee replacement in hospitals in canada . in the other case , mental health was measured by the geriatric depression scale gds in a longitudinal study of 150 older american adults . another general population study from germany found that in healthier individuals , positive affect was more important than physical functioning in predicting self rated general health , whereas among less healthy individuals the opposite was found . positive affect in that study was measured by the positive and negative affect schedule ( panas ) . the single question how would you rate your mental health ? was used in surveys much less than the single question about general health . ahmad et al . conducted a review of the literature and found fifty - seven studies that included a measurement of self rated mental health ( srmh ) but only four of these studies tried to validate the srmh against known measures of mental health . self - rated mental health was associated with use of mental health services and of medical care services . when checked against other mental health indicators the use of srmh as a proxy for mental morbidity was not recommended . flieshman and zuvekas examined the associations between srmh and measures of psychological distress , activity limitations and social role functioning . they found that the measures of emotional / psychological distress were correlated with each other much more strongly than they were with srmh , while srmh was related to physical health . their conclusion was that the self rated mental health was not a pure measure of mental morbidity and that it can not be used as the sole indicator of psychological distress in surveys . mawani and gilmour checked the associations between the srmh , mental health diagnoses , wmh - cidi diagnoses and measures of psychological distress . they found that a sizeable percentage of respondents who were classified as having mental disorders perceived their mental health as good ; on the other hand , respondents who did not pass the threshold for diagnoses perceived their mental health as fair / poor . their conclusion was that the srmh should not be used as a proxy for mental morbidity because it underestimates the prevalence of mental morbidity , and may reflect other factors as well . to our knowledge , there were no studies that checked simultaneously the self ratings of physical health , mental health and overall health . using data from a large representative sample of the adult population of israel , that included the three self rating scales as well as other objective health status indicators , we tried to understand what is the contribution of the self rating of mental health to the self rating of general health . the israel national health survey followed procedures established by the world mental health survey ( wmh ) of the world health organization ( who ) . the sample was extracted from israel s national population register and constituted non - institutionalized legal residents aged 21 and over . the sample was designed to reflect the population distribution by age , gender and three population sectors : arabs ; post-1990 jewish immigrants from the former soviet union ; and jews and others , including jewish immigrants from countries other than the former soviet union . the sample interviewed for the israel national health survey was weighted back to the total population to compensate for unequal selection probabilities resulting from disproportionate stratification , clustering effects , and non - response . the weights were adjusted to make sample totals conform to known population totals taken from reliable central bureau of statistics sources . on first personal contact with each potential survey respondent , the interviewer explained the survey and obtained verbal informed consent . overall , 73% of those contacted agreed to be interviewed ( 88% of arab - israelis and 71% of jewish - israelis ) . a total of 4859 face - to - face interviews were conducted in arabic , hebrew or russian at respondents homes from may 2003 to april 2004 . professional survey interviewers , who were trained and supervised by the central bureau of statistics , used a laptop computer and computer - assisted personal interview methods . the human subjects committee based at eitanim - kfar shaul hospital approved the survey and the field procedures in november 2000 . the diagnostic instrument used in the who - wmh was the composite international diagnostic interview ( cidi ) . the survey assessed for : anxiety disorders [ panic disorder , generalized anxiety disorder ( gad ) , agoraphobia without panic disorder , and post - traumatic stress disorder ( ptsd ) ] ; mood disorders ( major depressive disorder , dysthymia , bipolar i and ii disorders ) ; and substance abuse disorders ( alcohol abuse , alcohol dependency , drug abuse , drug dependency ) . the presence of a disorder was determined by whether respondents past or current symptoms met 30 days , 12-month or lifetime diagnostic criteria for a dsm iv disorder . for each disorder , when a respondent endorsed a specific screening item , he or she was asked all the questions in the cidi diagnostic section for that disorder to establish the presence of a current disorder . organic exclusion criteria were taken into account in the determination of the dsm - iv diagnoses . the validity of the who - wmh cidi as a diagnostic tool was assessed in france , italy , spain , and the united states by a clinical reappraisal study which found that the individual - level concordance between the scid and cidi for 12-month prevalence of any mood disorder , any anxiety disorder , and any disorder overall was substantial ( area under the curve in the range of 0.8 to 0.9 . for the analyses reported here , respondents were grouped into those with or without any mood or anxiety disorder , because of a large overlap between the two groups of diagnoses . the present analysis distinguished between respondents with and without mood or anxiety disorder in the 30 days before the interview . the conditions listed were heart attack , heart disease , stroke , high blood pressure , asthma , chronic obstructive pulmonary disorder , emphysema or other lung disease , tuberculosis , diabetes , kidney disease , neurological conditions , thyroid disease , cancer , chronic back or neck pain , arthritis or rheumatism , headaches , or any other chronic pain . in this analysis , respondents with chronic physical conditions were grouped into two categories : those reporting any one of the conditions and those reporting none . methodological research has shown that such checklists provide useful information about treated or currently untreated chronic conditions . the whodas is a disability scale that includes six scales : i ) role functioning , ii ) cognition , iii ) mobility , iv ) self - care , v ) social interaction and vi ) participation . the four questions that assessed role functioning were all frequency questions while other domains consist mostly of severity items . the calculation of the total score followed the method described in buist - bouwman : the resulting total score ranged between a score of 0 indicating no disability and 100 indicating maximum disability with intermediate scores indicating the percentage of the maximum possible score . in the present analysis , the total score was grouped into three categories : maximum disability = 100 , 9% of the entire sample , no disability = 0.47% of the entire sample and partial disability 0 > score < 100 , 44% of the entire sample . the rating of physical , mental and overall health were measured using the standard items how would you rate your . excellent , very good , good , fair , or poor ? since the connotation of good was neither very good nor bad , the present analysis followed others and grouped all three variables into two categories : positive excellent / very good and negative good / fair / poor . the control variables included the socio - demographic characteristics : age groups , gender , marital status , education , income and population groups [ classified as i ) arabs ii ) post-1990 jewish immigrants from the former soviet union iii ) jews and others , including jewish immigrants from countries other than the former soviet union ] . the data were weighted to adjust for the differential probabilities of respondents selection and non - response and for differences between the sample and the adult population in israel . cross tabulation of age groups by positive self rated physical health , positive self rated mental health , presence of chronic physical conditions and presence of any depression or anxiety disorders was done to show how these measures change with age . logistic regression analyses were conducted to estimate the association between dichotomous positive / negative outcomes of self rated health measures and the socio - demographic and health status indicators . estimates of odds ratios ( ors ) , the corresponding standard errors and 95% confidence intervals ( ci ) were also obtained from logistic regression using sudaan . the conditions listed were heart attack , heart disease , stroke , high blood pressure , asthma , chronic obstructive pulmonary disorder , emphysema or other lung disease , tuberculosis , diabetes , kidney disease , neurological conditions , thyroid disease , cancer , chronic back or neck pain , arthritis or rheumatism , headaches , or any other chronic pain . in this analysis , respondents with chronic physical conditions were grouped into two categories : those reporting any one of the conditions and those reporting none . methodological research has shown that such checklists provide useful information about treated or currently untreated chronic conditions . the whodas is a disability scale that includes six scales : i ) role functioning , ii ) cognition , iii ) mobility , iv ) self - care , v ) social interaction and vi ) participation . the four questions that assessed role functioning were all frequency questions while other domains consist mostly of severity items . the calculation of the total score followed the method described in buist - bouwman : the resulting total score ranged between a score of 0 indicating no disability and 100 indicating maximum disability with intermediate scores indicating the percentage of the maximum possible score . in the present analysis , the total score was grouped into three categories : maximum disability = 100 , 9% of the entire sample , no disability = 0.47% of the entire sample and partial disability 0 > score < 100 , 44% of the entire sample . the rating of physical , mental and overall health were measured using the standard items how would you rate your . excellent , very good , good , fair , or poor ? since the connotation of good was neither very good nor bad , the present analysis followed others and grouped all three variables into two categories : positive excellent / very good and negative good / fair / poor . the control variables included the socio - demographic characteristics : age groups , gender , marital status , education , income and population groups [ classified as i ) arabs ii ) post-1990 jewish immigrants from the former soviet union iii ) jews and others , including jewish immigrants from countries other than the former soviet union ] . the data were weighted to adjust for the differential probabilities of respondents selection and non - response and for differences between the sample and the adult population in israel . cross tabulation of age groups by positive self rated physical health , positive self rated mental health , presence of chronic physical conditions and presence of any depression or anxiety disorders was done to show how these measures change with age . logistic regression analyses were conducted to estimate the association between dichotomous positive / negative outcomes of self rated health measures and the socio - demographic and health status indicators . estimates of odds ratios ( ors ) , the corresponding standard errors and 95% confidence intervals ( ci ) were also obtained from logistic regression using sudaan . table 1 presents the distribution of the socio - demographic and health status indicators used in the analysis . we found that the 63% of the total sample rated their mental health as positive , 55% rated their overall health as positive but only 49% rated their physical health as positive . men rated their health consistently better than females : 66.5% of the males rated their mental health as positive compared to 61% of the females ( =14.7 ; p<0.001 ) , 52% of the males rated their physical health as positive compared to 48% of the females ( =11.8 ; p<0.01 ) and 58% of the males rated their overall health as positive compared to 53% of the females ( =11.2 ; p<0.01 ) . immigrants rated their health consistently lower ( 18% , 26% , 16% positive on the overall , mental and the physical health respectively ) than the arabs ( 55% , 61% , 54% ) and the jews and others ( 64% , 73% , 56% ) ( p<0.001 ) . those with high income rated their health consistently better ( 60% , 69% , 54% ) than those with low income ( 49% , 56% , 45% ) ( p<0.005 ) . those in the 21 - 34 age group rated their health consistently better ( 77% , 81% , 73% ) than those in the 65 + age group ( 18% , 35% , 13% ) ( p<0.001 ) . those who never married rated their health consistently better ( 75% , 78% , 71% ) than those who were divorced or separated ( 27% , 38% , 25% ) ( p=0.000 ) and those with high education rated their health consistently better ( 62% , 69% , 54% ) than those with low education ( 36% , 45% , 31% ) ( p<0.005 ) . table 2 presents the associations between health status indicators and the self rated physical health ( hsr ) and mental health ( mhsr ) . the odds ratios presented in the table were adjusted in the logistic regression for age , population groups , income , educational level and disability status . table 2 shows that self rated positive physical health is strongly related to chronic physical conditions but much less to mental disorders . likewise , self rated positive mental health is strongly related to mental disorders , but much less to chronic physical conditions . figure 1 shows how chronic physical conditions , mental disorders and the self rating scales of physical and mental health change with age . figure 1 shows that the percentage of those with chronic conditions goes up from 32% in the 21 - 35 age group to 78% in the 65 + group ( =653.9 ; p<0.001 ) while the poor self rated physical health follows the same pattern with a rise from 27% to 87% =1402.39 ; p<0.001 ) . on the other hand the poor self rated mental health goes up from 19.5% in the 21 - 35 age group to 64.8% in the 65 + group ( =609.98 ; p<0.001 ) while the percentage of those with any mental disorder does not change at all . table 3 presents the associations between self rated overall health ( osr ) and the self rated physical and mental health in two models : with and without the health status indicators . the odds ratios presented here were adjusted in the logistic regression for the variables population groups , age groups and disability level . table 3 shows that self rated mental and physical health are much stronger predictors of osr than the health status indicators of chronic conditions , mental disorders and disability . those with positive self rated mental health have 93 times the odds of reporting good positive overall health whereas those with positive self rated physical health have 40 times the odds of reporting positive overall health . after accounting for the self rated health measures hsr and mhsr , the predictive value of the health status indicators is much smaller : those with no mental disorders are six times more likely to have a positive osr , those with no disability have 2.5 times the odds of reporting positive osr and those with no chronic condition have only 1.33% times the odds of reporting positive osr . yet , the age group is still a significant predictor of osr beyond the self rating measures and the health status indicators . table 3 showed that after accounting for the main health variables , positive self rated mental health was twice as important as positive physical health in the prediction of positive overall health . the odds for positive overall health were 93 higher if the rating of mental health was positive compared to 40 times higher if the rating of physical health was positive . table 2 presents the associations between health status indicators and the self rated physical health ( hsr ) and mental health ( mhsr ) . the odds ratios presented in the table were adjusted in the logistic regression for age , population groups , income , educational level and disability status . table 2 shows that self rated positive physical health is strongly related to chronic physical conditions but much less to mental disorders . likewise , self rated positive mental health is strongly related to mental disorders , but much less to chronic physical conditions . figure 1 shows how chronic physical conditions , mental disorders and the self rating scales of physical and mental health change with age . figure 1 shows that the percentage of those with chronic conditions goes up from 32% in the 21 - 35 age group to 78% in the 65 + group ( =653.9 ; p<0.001 ) while the poor self rated physical health follows the same pattern with a rise from 27% to 87% =1402.39 ; p<0.001 ) . on the other hand the poor self rated mental health goes up from 19.5% in the 21 - 35 age group to 64.8% in the 65 + group ( =609.98 ; p<0.001 ) while the percentage of those with any mental disorder does not change at all . table 3 presents the associations between self rated overall health ( osr ) and the self rated physical and mental health in two models : with and without the health status indicators . the odds ratios presented here were adjusted in the logistic regression for the variables population groups , age groups and disability level . table 3 shows that self rated mental and physical health are much stronger predictors of osr than the health status indicators of chronic conditions , mental disorders and disability . those with positive self rated mental health have 93 times the odds of reporting good positive overall health whereas those with positive self rated physical health have 40 times the odds of reporting positive overall health . after accounting for the self rated health measures hsr and mhsr , the predictive value of the health status indicators is much smaller : those with no mental disorders are six times more likely to have a positive osr , those with no disability have 2.5 times the odds of reporting positive osr and those with no chronic condition have only 1.33% times the odds of reporting positive osr . yet , the age group is still a significant predictor of osr beyond the self rating measures and the health status indicators . table 3 showed that after accounting for the main health variables , positive self rated mental health was twice as important as positive physical health in the prediction of positive overall health . the odds for positive overall health were 93 higher if the rating of mental health was positive compared to 40 times higher if the rating of physical health was positive . data from the israel national health survey based on a representative sample of the adult population was used to investigate the differences between the three self rated measures of health . the general distribution of the self rated measures of health replicates results obtained in previous large population surveys showing that rates of positive self rated health are lower among females compared to males , that older individuals rate their health as less positive compared to younger individuals and that socio demographic variables such as education and income are related to the self rating of mental , physical and overall health . the main findings from the present study show that the three self rating scales represent three different domains of health : the self rating mental health and the self rating physical health maintain their distinctiveness : self rated mental health is not related to chronic physical conditions ( as does self rated physical health ) , and self rated physical health is not related to mental disorders ( as does self rated mental health ) . the self rating of physical health has a monotonic relationship with the objective indicator of physical health . the self rating of mental health does not have a monotonic relationship with the absence of mental illness . the good self rating of mental health goes down with age even though there is no age difference in mental disorders . the self rated physical health and the self rated mental health are both strong predictors of the self rated overall health , independent of the objective measures of health . this was shown when the odds of predicting the self rating of the overall health remained the same regardless of whether the objective measures of health were included in the regression equation . the self rated mental and physical health are 10 times stronger in predicting self rated overall health than any of the objective health indicators . the self rated mental health is two times more important than the self rated physical health in predicting the self rated overall health . the stronger weight of the self rated mental health implies that the mental component in the self rating of overall health is stronger than the physical one . this result reinforces conclusions of previous studies : in a cross - national survey of university students in germany , bulgaria , and poland , the overall self rated health status was assessed along with physical and psychological health as well as with social and socio demographic variables . that study found that psychosomatic complaints were the most important indicator in predicting the self rated health status . a similar finding was reported by ormel et al . from a study using a large sample of late middle - aged and older persons living independently . they found that the unique contribution of depressive symptoms on poor health perception outranked those of the medical conditions . in a survey with a follow - up period of 1 - 5 years found that those who were happier and more energetic at baseline rated their health better in the next 5 years , even after accounting for age , negative health indicators at baseline , disease status and functioning . moreover , positive indicators of functioning ; such as physical activity , work , and exercise ; did not significantly predict future self assessment of health . mental disorder was defined in our study by the presence of any mood or anxiety disorder in the past 30 days and its prevalence was the same in the four age groups . two major surveys checked the one year prevalence of any mood or anxiety and found it to be significantly less prevalent in older age groups . on the other hand , mental health which refers to hedonic well - being : feelings of happiness , satisfaction and interest in life , and to eudemonic well - being optimal functioning and self actualization , was more prevalent in younger age groups . in their review of studies on the concept of mental health , westerhof and keyes found that older age was correlated with lower positive affect , less feeling of personal growth and purpose in life , less meaning in life and less social coherence and social contribution . the empirical separation between mental health and mental illness was found also by weich et al . who used principal component analysis to describe the underlying factor structure of mental wellbeing . they found also that well - being and mental disorders are correlated but independent dimensions . the use of three separate self rating scales for mental health , physical health and overall health enabled us to observe the unique contribution of the mental health component to the self rating of overall health . further studies should investigate prospectively which of the three dimensions better predicts mortality , morbidity , hospitalizations or use of services . a key strength of this study was the utilization of a large representative sample of the entire adult population with a relatively high response rate . trends observed in this type of sample are more likely to appear in other large population samples . the main limitations of this study are that all the health information was self - reported and that the health indicators used may not have covered all the ill health domains . it is not clear whether a more comparative wording compared to your age group would have changed the clear age gradient that was found in this study . our results show a different age trajectory between mental health and mental illness . mental disorder was defined in our study by the presence of any mood or anxiety disorder in the past 30 days and two major surveys checked the one year prevalence of any mood or anxiety and found it to be significantly less prevalent in older age groups . on the other hand , mental health which refers to hedonic well - being : feelings of happiness , satisfaction and interest in life , and to eudemonic well - being optimal functioning and self actualization , was more prevalent in younger age groups . in their review of studies on the concept of mental health , westerhof and keyes found that older age was correlated with lower positive affect , less feeling of personal growth and purpose in life , less meaning in life and less social coherence and social contribution . the empirical separation between mental health and mental illness was found also by weich et al . who used principal component analysis to describe the underlying factor structure of mental wellbeing . they found also that well - being and mental disorders are correlated but independent dimensions . the use of three separate self rating scales for mental health , physical health and overall health enabled us to observe the unique contribution of the mental health component to the self rating of overall health . further studies should investigate prospectively which of the three dimensions better predicts mortality , morbidity , hospitalizations or use of services . a key strength of this study was the utilization of a large representative sample of the entire adult population with a relatively high response rate . trends observed in this type of sample are more likely to appear in other large population samples . the main limitations of this study are that all the health information was self - reported and that the health indicators used may not have covered all the ill health domains . it is not clear whether a more comparative wording compared to your age group would have changed the clear age gradient that was found in this study .

the management of the interim phase of a complete oral rehabilitation in patients with severely worn dentition is often challenging due to the loss of occlusal vertical dimension , loss of tooth structure , uneven wear of teeth creating an uneven plane of occlusion , and parafunctional habits . this case report describes the management of excessive tooth tissue loss in a 45 year old woman with a history of bruxism , esthetical complaints in anterior teeth , and impaired dental function due to reduced tooth height . the patient used occlusal splint for a month and than resection of the alveolar bone was performed on the vestibular sides of the maxillary anterior teeth , except the interdental alveolar crest . maxillary anterior teeth were restored with zirconia porcelain . feldspathic porcelain was chosen to restore remaining teeth in both jaws ; the patient also was given an occlusion guard to protect the restoration against future bruxism . regardless of the cause of occlusal instability , it is important that the restorative dentist should be able to recognize its signs such as tooth hypermobility , tooth wear , periodontal breakdown , occlusal dimpling , stress fractures , exostosis , muscle enlargement , and loss of posterior disclusion . when restoring the worn dentition , the clinician should bear in mind the five p s : proper planning prevents poor performance . backgroundunlike the widely used self rated health , the self rated mental health was found unsuitable as a proxy for mental illness . this paper analyses the relationships between the self ratings of physical health , mental health and overall health , and their association of with the objective indicators for physical and mental health.design and methodsthe study is a secondary analysis of data from a nationwide representative sample of the non - institutionalized adult residents of israel in 2003 that was collected via computer - assisted personal interview methods [ n=4859].resultsthe self rated physical health and the self rated mental health were strongly related to each other yet the self rated mental health was not related to chronic physical conditions and the self rated physical health was not related to mental disorders . in a multiple logistic regression analysis , those with positive self rated mental health had 93 times the odds of reporting positive overall health whereas those with positive self rated physical health had 40 times the odds of reporting positive overall health.conclusionsthe self rating of mental health presents a qualitatively different dimension from mental illness . the self rated mental health is two times more important than the self rated physical health in predicting the self rated overall healthsignificance for public healththe present study is an original study on the self rated physical , mental and overall health measures . because of the wide range of associations with other health indicators , and the simplicity with which they are collected , self - rated health measures are widely used in large population surveys.the present study questions the automatic assumption that the self rated mental health functions as a proxy measure of psychiatric morbidity , and suggests that the self rated mental health is more closely related to subjective well - being . the results show that self rated mental health predicts self rated general health better than self rated physical health .